Antibody drug conjugates (adc) that bind to 161p2f10b proteins

ABSTRACT

Antibody drug conjugates (ADC&#39;s) that bind to 161P2F10B protein are described herein. 161P2F10B exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, the ADC&#39;s of the invention provide a therapeutic composition for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/302,489, filed on Feb. 8, 2010. The contents of thatapplication are hereby incorporated by reference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582006231Seqlist.txt Jan. 27,2011 43,609 bytes

FIELD OF THE INVENTION

The invention described herein relates to antibody drug conjugates(ADCs) thereof that bind proteins, termed 161P2F10B. The inventionfurther relates to prognostic, prophylactic and therapeutic methods andcompositions useful in the treatment of cancers.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

While previously identified markers such as PSA, PSM, PCTA and 161P2F10Bhave facilitated efforts to diagnose and treat prostate cancer, there isneed for the identification of additional markers and therapeutictargets for prostate and related cancers in order to further improvediagnosis and therapy.

Renal Cell Cancer (RCC) presently ranks 10th as the leading cause ofcancer death in the United States. An estimated 51,190 people will bediagnosed annually with renal cell carcinoma in the US and approximately12,890 died from the disease in 2007 (American Cancer Society).Historically, treatment has focused primarily on nephrectomy, followedby nonspecific immunotherapy, and sometimes radiation therapy (Hauke,2006). Nonspecific immunotherapy includes treatment with the cytokinesinterleukin-2 or interferon-α as either single agents or in combination.After surgical excision, 20-30% of patients will develop metastaticdisease within 1-3 years, often in the lung (Motzer, et al., 2006).Median survival for patients with metastatic disease is approximately 13months (Cohen and McGovern, 2005).

Since 2005, six (6) agents have been approved by FDA for the treatmentadvanced renal cell cancer. These advances include several agents thattarget the specific pathways implicated in renal cell cancer. Theseagents include sorafenib (Nexavar®, FDA approved in December 2005),sunitinib (Sutent®, FDA approved in January 2006), temsirolimus(Torisel®, FDA approved in May 2007), everolimus (Affinitor®, FDAapproved in March, 2009), bevacizumab (Avastin® in combination withinterferon alpha, FDA approved in August 2009) and pazopanib (Votrient®FDA approved in October 2009). However, despite advances in thetreatment, metastatic Renal cell cancer remains incurable and onlytemsirolimus was approved based on an advantage in overall survival.

Additionally, hepatocellular carcinoma (i.e., cancer of the liver)accounts for 80-90% of all liver cancers. This type of liver canceroccurs more often in men than in women. It is usually seen in peopleages 50-60. Generally, treatment of liver cancer is aggressive surgeryor a liver transplant which may successfully treat small or slow growingtumors if they are diagnosed early. However, few patients are diagnosedearly. Chemotherapy and radiation treatments are not usually effective.However, these therapies are used to shrink tumors so surgery has agreater chance of success. Sorafenib tosylate (Nexavar®) is nowavailable for patients with liver cancer. The prognosis for patientswith liver cancer is usually poor, since only 10-20% of hepatocellularcarcinomas can be removed using surgery. Accordingly, there is a need todevelop an agent used to treat liver cancer.

The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler andC. Milstein, Nature 256:495-497 (1975)) is being realized. Monoclonalantibodies have now been approved as therapies in transplantation,cancer, infectious disease, cardiovascular disease and inflammation.Different isotypes have different effector functions. Such differencesin function are reflected in distinct 3-dimensional structures for thevarious immunoglobulin isotypes (P. M. Alzari, et al., Annual Rev.Immunol., 6:555-580 (1988)).

Because mice are convenient for immunization and recognize most humanantigens as foreign, mAbs against human targets with therapeuticpotential have typically been of murine origin. However, murine mAbshave inherent disadvantages as human therapeutics. They require morefrequent dosing as mAbs have a shorter circulating half-life in humansthan human antibodies. More critically, the repeated administration ofmurine antibodies to the human immune system causes the human immunesystem to respond by recognizing the mouse protein as a foreign andgenerating a human anti-mouse antibody (HAMA) response. Such a HAMAresponse may result in allergic reaction and the rapid clearing of themurine antibody from the system thereby rendering the treatment bymurine antibody useless. To avoid such affects, attempts to create humanimmune systems within mice have been attempted.

Initial attempts hoped to create transgenic mice capable of respondingto antigens with antibodies having human sequences (See Bruggemann, etal., Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)), but were limitedby the amount of DNA that could be stably maintained by availablecloning vehicles. The use of yeast artificial chromosome (YAC) cloningvectors led the way to introducing large germline fragments of human Iglocus into transgenic mammals. Essentially a majority of the human V, D,and J region genes arranged with the same spacing found in the humangenome and the human constant regions were introduced into mice usingYACs. One such transgenic mouse strain is known as XenoMouse® mice andis commercially available from Amgen Fremont, Inc. (Fremont Calif.).

SUMMARY OF THE INVENTION

The invention provides antibody drug conjugates (ADCs) that bind to161P2F10B proteins. In some embodiments, the invention comprises fullyhuman antibodies conjugated with a therapeutic agent.

The invention further provides various immunogenic or therapeuticcompositions, such as antibody drug conjugates, and strategies fortreating cancers such as cancers of tissues listed in Table I.

The present invention relates to:

[1] An antibody drug conjugate comprising an antibody or antigen bindingfragment thereof that binds specifically to a 161P2F10B proteincomprising the amino acid sequence of SEQ ID NO:2, and wherein theantibody comprises the amino acid sequence of the V_(H) region of SEQ IDNO:7, from 20 to 142 and the V_(L) region of SEQ ID NO:8, from 20 to 127and wherein said antibody is conjugated to monomethyl auristatin F(MMAF).

[2] The antibody drug conjugate of [1], wherein the antigen bindingfragment is an Fab, F(ab′)₂ or Fv fragment.

[3] The antibody drug conjugate of [1], wherein the antibody is a fullyhuman antibody.

[4] The antibody drug conjugate of [1], which is recombinantly produced.

[5] A pharmaceutical composition that comprises the antibody drugconjugate of [1] in a human unit dose form.

[6] The pharmaceutical composition of [5], wherein the composition isfor cancer treatment.

[7] The pharmaceutical composition of [6], wherein the cancer is renalcancer or liver cancer.

[8] A method of inhibiting growth of cancer cells in a subject,comprising: administering to said subject an antibody drug conjugate of[1].

[9] A method of delivering a cytotoxic agent or a diagnostic agent to acell, comprising:

providing MMAF(s) conjugated to an antibody or antigen binding fragmentthereof that binds specifically to a 161P2F10B protein comprising theamino acid sequence of SEQ ID NO:2, and wherein the antibody comprisesthe amino acid sequence of the V_(H) region of SEQ ID NO:7, from 20 to142 and the V_(L) region of SEQ ID NO:8, from 20 to 127, to form anantibody drug conjugate; and,

exposing the cell to the antibody drug or fragment drug conjugate.

[10] A method for treating tumor in a mammal comprising treating themammal with an effective amount of an antibody drug conjugate of [1].

[11] A method for reducing tumor growth in a mammal comprising treatingthe mammal with an effective amount of a combination of an antibody drugconjugate of [1] and radiation.

[12] A method for reducing tumor growth in a mammal comprising treatingthe mammal with an effective amount of a combination of an antibody drugconjugate of [1] and a chemotherapeutic agent.

[13] A method for reducing tumor growth in a mammal comprising treatingthe mammal with an effective amount of a combination of an antibody drugconjugate of [1] and a drug or biologically active therapy.

[14] A method for treating cancer in a mammal, comprising treating themammal with an effective amount of a combination of an antibody drugconjugate of [1] and a chemotherapeutic agent.

[15] An antibody drug conjugate (ADC), wherein the ADC having theformula L-(LU-D)p, wherein: (a) L is the antibody comprising an antibodyor antigen binding fragment thereof that binds specifically to a161P2F10B protein comprising the amino acid sequence of SEQ ID NO:2, andwherein the antibody comprises the amino acid sequence of the V_(H)region of SEQ ID NO:7, from 20 to 142 and the V_(L) region of SEQ IDNO:8, from 20 to 127; (b) LU is a linker; (c) D is a drug moiety whereindrug is monomethyl auristatin F (MMAF); (d) p is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12.

[16] An antibody drug conjugate (ADC), wherein the ADC having thefollowing formula, wherein Ab-S is the antibody comprising an antibodyor antigen binding fragment thereof that binds specifically to a161P2F10B protein comprising the amino acid sequence of SEQ ID NO:2, andwherein the antibody comprises the amino acid sequence of the V_(H)region of SEQ ID NO:7, from 20 to 142 and the V_(L) region of SEQ IDNO:8, from 20 to 127; p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[17] An antibody drug conjugate comprising an antibody that bindsspecifically to a 161P2F10B protein comprising the amino acid sequenceof SEQ ID NO:2, and wherein the antibody comprises the amino acidsequence of the heavy chain of SEQ ID NO:7, from 20 to 468 and the lightchain of SEQ ID NO:8, from 20 to 233 and wherein said antibody isconjugated to monomethyl auristatin F (MMAF).

[18] An antibody drug conjugate (ADC), wherein the ADC having theformula L-(LU-D)p, wherein: (a) L is the antibody comprising an antibodythat binds specifically to a 161P2F10B protein comprising the amino acidsequence of SEQ ID NO:2, and wherein the antibody comprises the aminoacid sequence of the heavy chain of SEQ ID NO:7, from 20 to 468 and thelight chain of SEQ ID NO:8, from 20 to 233; (b) LU is a linker; (c) D isa drug moiety wherein drug is monomethyl auristatin F (MMAF); (d) p is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[19] An antibody drug conjugate (ADC), wherein the ADC having thefollowing formula, wherein Ab-S is the antibody comprising an antibodythat binds specifically to a 161P2F10B protein comprising the amino acidsequence of SEQ ID NO:2, and wherein the antibody comprises the aminoacid sequence of the heavy chain of SEQ ID NO:7, from 20 to 468 and thelight chain of SEQ ID NO:8, from 20 to 233; p is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The cDNA and amino acid sequence of 161P2F10B is shown inFIG. 1. The open reading frame extends from nucleic acid 44-2671including the stop codon.

FIG. 2. Nucleic Acid and Amino Acid sequences of 161P2F10B antibodies.

FIG. 2A. The cDNA and amino acid sequence of H16-7.8 heavy chain.Underlined is the leader sequence and double-underlined is the heavychain variable region. Dashed underline shows the human IgG2 constantregion.

FIG. 2B. The cDNA and amino acid sequence of H16-7.8 light chain.Underlined is the leader sequence and double-underlined is the lightchain variable region. Dashed underline shows the human Ig kappaconstant region.

FIG. 3. Amino Acid sequences of 161P2F10B antibodies.

FIG. 3A. The amino acid sequence of H16-7.8 heavy chain. Underlined isthe leader sequence and double-underlined is the heavy chain variableregion. Dashed underline shows the human IgG2 constant region.

FIG. 3B. The amino acid sequence of H16-7.8 light chain. Underlined isthe leader sequence and double-underlined is the light chain variableregion. Dashed underline shows the human Ig kappa constant region.

FIG. 4. Alignment of H16-7.8 antibodies to human germline.

FIG. 4A. Alignment of H16-7.8 heavy chain to human germlineVH4-31/D5-12/JH6.

FIG. 4B. Alignment of H16-7.8 light chain to human germline A26/JK1.

FIG. 5. H16-7.8 Recombinant Expression in CHO Cells. H16-7.8 heavy andlight chain sequences were cloned into expression vectors. Vectors weretransfected into CHO cells. 3T3-control and 3T3-161P2F10B cells werestained with H16-7.8 from either hybridoma or from CHO cells. Bindingwas detected by flow cytometry. Results show H16-7.8 recombinantlyexpressed in CHO cells is secreted and binds specifically tocell-surface 161P2F10B.

FIG. 6. Cell Binding and Affinity of H16-7.8 and H16-7.8mcMMAF. H16-7.8and H16-7.8mcMMAF were tested for the binding affinity to 161P2F10Bendogenously expressed on Ku812 cells. Briefly, eleven (11) dilutions ofH16-7.8 or H16-7.8mcMMAF were incubated with Ku812 cells (50,000 cellsper well) overnight at 4° C. at a final concentration of 160 nM to0.0001 nM. At the end of the incubation, cells were washed and incubatedwith anti-hIgG-PE detection antibody for 45 min at 4° C. After washingthe unbound detection antibodies, the cells were analyzed by FACS. MeanFlorescence Intensity (MFI) values are obtained (See, Table IV). MFIvalues were entered into Graphpad Prisim software and analyzed using theone site binding (hyperbola) equation of Y=Bmax*X/(Kd+X) to generateH16-7.8 or H16-7.8mcMMAF saturation curves shown in FIG. 6. Bmax is theMFI value at maximal binding of H16-7.8 or H16-7.8mcMMAF to 161P2F10B;Kd is H16-7.8 or H16-7.8mcMMAF binding affinity which is theconcentration of H16-7.8 or H16-7.8mcMMAF required to reach half-maximalbinding. The calculated affinity (Kd) of H16-7.8 and H16-7.8mcMMAF is0.06 nM and 0.19 nM, respectively on 161P2F10B endogenously expressed onthe surface of Ku812 cells.

FIG. 7. Binding of H16-7.8 and H16-7.8mcMMAF to Renal Cancer Cells.Human UGK-3 cells (patient derived clear cell renal cancer) and RXF-393cells (clear cell renal cancer) were stained with 10 μg/ml of nativeH16-7.8, H16-7.8mcMMAF, or an isotype control human IgG2 and evaluatedby FACS. The results in FIG. 7 (left panels) demonstrate strong stainingof the two different renal tumor cells with H16-7.8 (gray lines), butnot with the control MAb (filled histograms). The panels on the rightdemonstrate a similar strong staining of the same renal tumor cells withH16-7.8mcMMAF (gray lines). (FIG. 7; right panels). These results showthat both H16-7.8 and H16-7.8mcMMAF bind native 161P2F10B antigenexpressed on the surface of human cancer cells. Conjugation of nativeH16-7.8 to generate the H16-7.8mcMMAF did not alter its cell surfacebinding to native 161P2F10B antigen expressed on human cancer cells.

FIG. 8. Cell Cytotoxicity by H16-7.8mcMMAF. 2000 viable KU812 cells wereplated in triplicate on Day 0 and allowed to recover overnight. The nextday, serial 1:4 dilutions of different lots of H16-7.8mcMMAF or acontrol MAb conjugated with mcMMAF were added to yield the finalconcentrations. The cells were allowed to incubate for six (6) days atwhich time 20 μl of Alamar blue was added to each well. The plates wereincubated for an additional four (4) hours and the fluorescenceintensity read on a fluorescent plate reader using an excitationwavelength of 540 nM and an emission wavelength of 620 nM. The resultsshow that both lots of H16-7.8mcMMAF potently inhibited theproliferation of KU812 cells. The IC 50 was determined to be 0.2 nM and0.1 nM for Lots (1) and Lot (2) respectively. A fully human Control MAbthat does not bind KU812 cells was conjugated with mcMMAF to yield a DARof 3.9 (+/−0.2). The Control ADC (Control mcF) did not inhibit KU812cell proliferation further demonstrating the specificity ofcytotoxicity. Thus, these results indicate that H16-7.8mcMMAF canselectively deliver a cytotoxic drug to 161P2F10B expressing cells,leading to their killing.

FIG. 9. Efficacy of H16-7.8mcMMAF in subcutaneously established humanrenal cancer xenograft UG-K3 in SCID mice. In this experiment,patient-derived human renal cancer xenograft UG-K3 was maintained byserial passages in SCID mice. Stock tumors were harvested sterilely andminced to small pieces. The tumor pieces were enzymatically digested tosingle cell suspensions using Liberase Blendzyme (Roche Applied Science,Indianapolis, Ind.). 1.5×10⁶ cells were injected into the flanks ofindividual SCID mice and tumors were allowed to grow untreated untilthey reached an approximate volume of 100 mm³. Animals were randomlyassigned to the following cohorts: an H16-7.8mcMMAF treated group, anH16-7.8 control and a 5% Dextrose control. H16-7.8mcMMAF and H16-7.8were dosed at 10 mg/kg once on day 0 by intravenous bolus injection. Theamount of H16-7.8mcMMAF and H16-7.8 administered was based on theindividual body weight of each animal obtained immediately prior todosing. The 5% Dextrose control was dosed at 150 μL per animal. Tumorgrowth was monitored using caliper measurements every 3 to 4 days untilthe end of the study. Tumor volume is calculated as Width²×Length/2,where width is the smallest dimension and length is the largest. Animalsin control groups were humanely euthanized when tumors reachedapproximately 1000 mm³. Animals in H16-7.8mcMMAF treated group weremonitored for an additional two weeks before sacrifice. Statisticalanalysis was performed at the last time point when data for both controlgroups were available, using Kruskal-Wallis test with α=0.05.

The results demonstrated that treatment of UG-K3 renal clear cellxenograft tumors with H16-7.8mcMMAF at all doses and schedules examinedresulted in significant inhibition of tumor growth in SCID mice.

FIG. 10. Growth Inhibition of Established Orthotopic UG-K3 Xenografts byH16-7.8mcMMAF. The ability of H16-7.8mcMMAF to inhibit the growth ofestablished renal tumors grown orthotopically was evaluated usingpatient-derived, UG-K3 tumor xenografts. Briefly, stocks of UG-K3 tumorswere digested enzymatically and 1.5 million viable cells were surgicallyimplanted into the kidneys of male SCID mice on Day 0. The tumors wereallowed to grow for 7 days at which time animals were randomized to 4different treatment groups (n=10 per group). Animals randomized to GroupA received Control ADC at 5 mpk, Group B received H16-7.8mcMMAF at 3mg/kg and Group C received H16-7.8mcMMAF at 5 mg/kg administered every 4days for a total of 4 doses. Group D received H16-7.8mcMMAF at 10 mg/kgone time. At the end of the study (Day 41) the animals were sacrificedand the right and left kidneys weighed on an electronic balance. Thetumor weights plotted on the graph were determined by subtracting theweight of the tumor-free contralateral kidney from the weight of thetumor-bearing right kidney.

The results demonstrated that treatment of UG-K3 renal clear cellxenograft tumors with H16-7.8mcMMAF at all doses and schedules examinedresulted in dramatic inhibition of tumor growth. Tumor weights in allH16-7.8mcMMAF treatment groups (B, C, and D) were less than 1% of thetumor weights in the Control treated group. These differences werehighly statistically significant (p<0.0001, ANOVA).

FIG. 11. Efficacy of H16-7.8mcMMAF in subcutaneously established humanrenal cancer xenograft RXF-393 in SCID mice. In this experiment, humanrenal cancer cells RXF-393 (0.5×10⁶ cells per mouse) were injected intothe flanks of individual mice and tumors were allowed to grow untreateduntil they reached an approximate volume of 100 mm³. Animals were thenrandomly assigned to the following cohorts: an H16-7.8mcMMAF treatedgroup, an H16-7.8 treated group and a 5% Dextrose control. H16-7.8mcMMAFand H16-7.8 were dosed at 10 mg/kg once a week for a total of two dosesby intravenous bolus injection. The amount of H16-7.8mcMMAF and H16-7.8administered was based on the individual body weight of each animalobtained immediately prior to dosing. The 5% Dextrose control was dosedat 150 μL per animal. Tumor growth was monitored using calipermeasurements every 3 to 4 days until the end of the study. Tumor volumeis calculated as Width²×Length/2, where width is the smallest dimensionand length is the largest. Animals in control groups were humanelyeuthanized when tumors reached approximately 1000 mm³ Animals inH16-7.8mcMMAF treated group were monitored for an additional two weeksbefore sacrifice.

The results demonstrated that treatment of RFX-393 human renal cancerxenograft tumors with H16-7.8mcMMAF at all doses and schedules examinedresulted in significant inhibition of tumor growth in SCID mice.Statistical analysis was performed at the last time point when data inboth control groups were available, using Kruskal-Wallis test withα=0.05.

FIG. 12. Efficacy Study of H16-7.8 compared to H16-7.8mcMMAF insubcutaneously established human renal cancer SKRC-01 in SCID Mice.Human renal cancer cells SKRC-01 (0.8×10⁶ cells per mouse) were injectedinto the flanks of individual mice. Tumors were allowed to growuntreated until they reached an approximate volume of 100 mm³. On day 0when tumors reach 100 mm³, animals were randomly assigned to thefollowing cohorts: an H16-7.8mcMMAF treated group, an H16-7.8 treatedgroup and a 5% Dextrose control. H16-7.8mcMMAF and H16-7.8 were dosed at4 mg/kg every four days for a total of four doses by intravenous bolusinjection. The amount of H16-7.8mcMMAF and H16-7.8 administered wasbased on the individual body weight of each animal obtained immediatelyprior to dosing. The 5% Dextrose control was dosed at 150 μL per animal.Tumor growth was monitored using caliper measurements every 3 to 4 days.Tumor volume was calculated as Width²×Length/2, where width is thesmallest dimension and length is the largest.

The results show that the ADC H16-7.8mcMMAF significantly inhibited thegrowth of SKRC-01 tumor formation while the naked MAb H16-7.8 had noeffect. Thus, the ADC H16-7.8mcMMAF had a significantly more prominenteffect that the naked antibody H16-7.8.

FIG. 13. Efficacy Study of H16-7.8mcMMAF compared to other 161P2F10BAntibody Drug Conjugates (ADCs) in subcutaneous established UG-K3 inSCID mice. In another experiment, human renal cancer cells UG-K3(1.5×10⁶ cells per mouse) were injected into the flanks of individualmice. Tumors were allowed to grow untreated until they reached anapproximate volume of 100 mm³. On day 0 when tumors reach 100 mm³,animals were randomly assigned to the following cohorts: anH16-7.8mcMMAF, an H16-7.8vcMMAE, and H16-1.11mcMMAF, and H16-1.11vcMMAE,a PBS control, and a control MAb-vcMMAE treated group. All antibody drugconjugates (ADCs) were dosed at 10 mg/kg once on day 0. The amount ofeach ADC administered was based on the individual body weight of eachanimal obtained immediately prior to dosing. The PBS control was dosedat 150 μ/L per animal. Tumor growth was monitored using calipermeasurements every 3 to 4 days. Tumor volume was calculated asWidth²×Length/2, where width is the smallest dimension and length is thelargest.

The results show that the ADCs H16-7.8vcMMAE and H16-1.11vcMMAE did notinhibit tumor formation growth. Additionally, both the H16-7.8mcMMAF andH16-1.11mcMMAF significantly inhibited the growth of UG-K3 tumorformation during the first thirty (30) days. After day thirty (30) theH16-7.8mcMMAF had a significantly more prominent effect when compared toH16-1.11mcMMAF.

FIG. 14. Peptide maps of H16-7.8mcMMAF and H16-7.8. The obtainedH16-7.8mcMMAF and H16-7.8 were treated with dithiothreitol (DTT) toreduce disulfide bonds, followed by alkylation of the resulting freecysteines. Guanidine was used in this step to ensure completedenaturation of the protein. After dialysis to remove the guanidine, thesamples were digested with a specific endoproteinase, Lys-C. Lys-Ccleaves peptide bonds on the C-terminal side of lysine residues. Theresulting peptides were analyzed by reversed phase chromatographycoupled to mass spectrometry. The reversed phase retention times and theobserved mass to charge ratios of the peaks were compared betweenH16-7.8mcMMAF and H16-7.8. LC-MS (liquid chromatography-massspectrometry) analysis was carried out using a WATERS Acquity UPLCcoupled to a WATERS Q-TOFp mass spectrometer. The digested sample wasapplied to YMC C18 column and eluted with an acetonitrile gradientcontaining trifluoroacetic acid. The results show, peak intensitiesindicated by asterisk were reduced in the conjugated antibody comparedto the native antibody. The peaks marked with an arrow represent newpeaks that appeared on the conjugated antibody peptide map.Specifically, the peaks marked with either an asterisk or with an arroware believed to be a peptide destined for conjugation and the resultingconjugated peptide, respectively.

FIG. 15. Mass spectra of the (*) peak. The results show a portion of themass spectra of the peak marked with an asterisk in FIG. 14. The massvalue of the signal that changed during conjugation is indicated by the“plus” sign. This peptide with an approximate m/z of 970.4 (+3 chargestate) was identified as C225-K250 that originated from the hinge regionof the heavy chain and contains the expected conjugation sites.

FIG. 16. Extracted ion chromatograms (XIC) of MSE on peptide maps forH16-7.8mcMMAF and H16-7.8 at 619.4 m/z. In order to identify the newlyappeared peaks which are believed to be conjugated peptide in FIG. 14above, LC-MS analysis was conducted using the elevated-energy (MSE) dataacquisition technique. This Figure shows the extracted ion chromatograms(XIC) for peptide maps of H16-7.8mcMMAF and H16-7.8 using the m/z of619.4. This ion corresponds to a fragment ion of the drug moiety. Peaksobserved in XIC at 619.4 are almost identical to the peaks marked withan arrow in FIG. 14. Furthermore, no such peaks were detected in thechromatogram of the native antibody. These observations suggest that thedetected peaks in the XIC at m/z of 619.4 were apparently drugconjugated peptides and are identified by its intact mass values. Theresult was summarized in Table V. These results suggest that in case ofthe conjugate, predominant peptides are those conjugated to 2 drugs onthe hinge region of heavy chain. These data are consistent with the dataobtained by the other orthogonal such as a DAR analysis.

FIG. 17. Example Spectra showing mass profiles of deglycosylatedH16-7.8mcMMAF ADC. The full mass of the deglycosylated H16-7.8mcMMAF wasdetermined by electrospray ionization time-of-flight (ESI-TOF) massspectrometry. Test samples were diluted by 250 mM sodium phosphatebuffer, pH 7.5 and then incubated overnight at 37° C. withglycopeptidase F. The samples were injected onto a PLRPTM column (VarianTechnology), equilibrated at 90° C., and eluted with anacetonitrile/water gradient. The sample peaks were analyzed by anAcquity UPLC system coupled to a WATERS Synapt mass spectrometer(Waters) and masses were reconstructed from the raw data by a MaxEnt1software. An example mass spectral profile for the deglycosylatedH16-7.8mcMMAF is shown. The predominant drug conjugated antibody was a4-drug loading species. This observation including an abundance of theunconjugated antibody in H16-7.8mcMMAF was consistent with the resultsobtained by the other orthogonal methods, such as DAR by RP-HPLC,peptide mapping, and HIC assay.

FIG. 18. Drug Antibody Ratio (DAR) profile of H16-7.8mcMMAF. DARanalysis was conducted for quantitative HPLC determination of therelative amount of drug loading in each Light chain and Heavy chain. DARanalyses were carried out using a PLRP-S analytical column, 2.1 mm×50mm, with mobile phase A consisting of 2.0% formic acid and mobile phaseB consisting of 2.0% formic acid plus 90% acetonitrile. A representativeDAR profile for H16-7.8mcMMAF is shown. DAR value is 4.0. The sample wassubjected to LC-MS analysis using same HPLC conditions of this method toidentify the observed peak. Results are summarized in Table VI. The peakidentification of the DAR results obtained during the qualification ofthis method has been confirmed orthogonally by LC-MS.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

I.) Definitions

II.) 161P2F10B Antibodies

III.) Antibody Drug Conjugates Generally

-   -   III(A). Auristatins and Dolostatins

IV.) Antibody Drug Conjugates which Bind 161P2F10B

V.) Linker Units

VI.) The Stretcher Unit

VII.) The Amino Acid Unit

VIII.) The Spacer Unit

IX.) The Drug Unit

X.) Drug Loading

XI.) Methods of Determining Cytotoxic effect of ADCs

XII.) Treatment of Cancer(s)

XIII.) 161P2F10B as a Target for Antibody-based Therapy

XIV.) 161P2F10B ADC Cocktails

XV.) Combination Therapy

XVI.) KITS/Articles of Manufacture

I.) Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook, et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

The terms “advanced cancer”, “locally advanced cancer”, “advanceddisease” and “locally advanced disease” mean cancers that have extendedthrough the relevant tissue capsule, and are meant to include stage Cdisease under the American Urological Association (AUA) system, stageC1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+disease under the TNM (tumor, node, metastasis) system. In general,surgery is not recommended for patients with locally advanced disease,and these patients have substantially less favorable outcomes comparedto patients having clinically localized (organ-confined) cancer.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine(see Formula XVI infra).

The abbreviation “MMAE” refers to monomethyl auristatin E (see FormulaXI infra).

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid (see Formula XX infra).

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid (see Formula XXI infra).

The abbreviation “MMAF” refers todovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula XVIVinfra).

Unless otherwise noted, the term “alkyl” refers to a saturated straightor branched hydrocarbon having from about 1 to about 20 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 8 carbon atomsbeing preferred. Examples of alkyl groups are methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, Cert-butyl, n-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl, and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groupscan be optionally further substituted with one or more groups including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂ , —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃ , —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the terms “alkenyl” and “alkynyl” refer tostraight and branched carbon chains having from about 2 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 2 to about 8carbon atoms being preferred. An alkenyl chain has at least one doublebond in the chain and an alkynyl chain has at least one triple bond inthe chain. Examples of alkenyl groups include, but are not limited to,ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl,-1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are notlimited to, acetylenic, propargyl, acetylenyl, propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,can be optionally substituted with one or more groups, preferably 1 to 3groups (and any additional substituents selected from halogen),including but not limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, or -aryl and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈alkynyl groups can be optionally further substituted with one or moresubstituents including, but not limited to, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂,—C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH,—N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl.

Unless otherwise noted, the term “alkylene” refers to a saturatedbranched or straight chain hydrocarbon radical having from about 1 toabout 20 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), with from about 1to about 8 carbon atoms being preferred and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkylenesinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene,1,4-cyclohexylene, and the like. Alkylene groups, whether alone or aspart of another group, can be optionally substituted with one or moregroups, preferably 1 to 3 groups (and any additional substituentsselected from halogen), including, but not limited to, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN, where each R′ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl and wherein said—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groups can be furtheroptionally substituted with one or more substituents including, but notlimited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “alkenylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon doublebond. Exemplary alkenylene groups include, for example, ethenylene(—CH═CH—) and propenylene (—CH═CHCH₂—).

Unless otherwise noted, the term “alkynylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon triplebond. Exemplary alkynylene groups include, for example, acetylene(—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

Unless otherwise noted, the term “aryl” refers to a monovalent aromatichydrocarbon radical of 6-20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)derived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Some aryl groups are represented in theexemplary structures as “Ar”. Typical aryl groups include, but are notlimited to, radicals derived from benzene, substituted benzene, phenyl,naphthalene, anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NO₂, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryland wherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂ , —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “arylene” refers to an optionallysubstituted aryl group which is divalent (i.e., derived by the removalof two hydrogen atoms from the same or two different carbon atoms of aparent aromatic ring system) and can be in the ortho, meta, or paraconfigurations as shown in the following structures with phenyl as theexemplary aryl group.

Typical “—(C₁-C₈ alkylene)aryl,” “—(C₂-C₈ alkenylene)aryl”, “and —(C₂-C₈alkynylene)aryl” groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

Unless otherwise noted, the term “heterocycle,” refers to a monocyclic,bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (alsoreferred to as ring members) wherein at least one ring atom in at leastone ring is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocyclicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S),and a bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N,O, P, or S). The ring that includes the heteroatom can be aromatic ornon-aromatic. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure.

Heterocycles are described in Paquette, “Principles of ModemHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.82:5566 (1960).

Examples of “heterocycle” groups include by way of example and notlimitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl),thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl,indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl,4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl. Preferred “heterocycle” groups include, but are notlimited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl.

A heterocycle group, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 2groups, including but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryland wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, and -aryl groupscan be further optionally substituted with one or more substituentsincluding, but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂ , —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected from—H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

By way of example and not limitation, carbon-bonded heterocycles can bebonded at the following positions: position 2, 3, 4, 5, or 6 of apyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole orthiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole;position 2 or 3 of an aziridine; position 2, 3, or 4 of an azetidine;position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline. Still more typically, carbon bondedheterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl,6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl,3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole;position 2 of a isoindole, or isoindoline; position 4 of a morpholine;and position 9 of a carbazole, or β-carboline. Still more typically,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

Unless otherwise noted, the term “carbocycle,” refers to a saturated orunsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring systemhaving from 3 to 14 ring atoms (and all combinations and subcombinationsof ranges and specific numbers of carbon atoms therein) wherein all ofthe ring atoms are carbon atoms. Monocyclic carbocycles preferably have3 to 6 ring atoms, still more preferably 5 or 6 ring atoms. Bicycliccarbocycles preferably have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. The term “carbocycle”includes, for example, a monocyclic carbocycle ring fused to an arylring (e.g., a monocyclic carbocycle ring fused to a benzene ring).Carbocyles preferably have 3 to 8 carbon ring atoms.

Carbocycle groups, whether alone or as part of another group, can beoptionally substituted with, for example, one or more groups, preferably1 or 2 groups (and any additional substituents selected from halogen),including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independently selectedfrom —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl andwherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Examples of monocyclic carbocylic substituents include -cyclopropyl,-cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl,-1-cyclopent-3-enyl, cyclohexyl, -1-cyclohex-1-enyl, -1-cyclohex-2-enyl,-1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl, -1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

A “carbocyclo,” whether used alone or as part of another group, refersto an optionally substituted carbocycle group as defined above that isdivalent (i.e., derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent carbocyclic ring system).

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₈alkylene)aryl” or “—C₁-C₈ alkylene(aryl)” refers to a C₁-C₈ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atoms bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.The group can, however, generally have any number of substituentsselected from halogen. Groups that are substituted are so indicated.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

Protective groups as used herein refer to groups which selectivelyblock, either temporarily or permanently, one reactive site in amultifunctional compound. Suitable hydroxy-protecting groups for use inthe present invention are pharmaceutically acceptable and may or may notneed to be cleaved from the parent compound after administration to asubject in order for the compound to be active. Cleavage is throughnormal metabolic processes within the body. Hydroxy protecting groupsare well known in the art, see, Protective Groups in Organic Synthesisby T. W. Greene and P. G. M. Wuts (John Wiley & sons, 3^(rd) Edition)incorporated herein by reference in its entirety and for all purposesand include, for example, ether (e.g., alkyl ethers and silyl ethersincluding, for example, dialkylsilylether, trialkylsilylether,dialkylalkoxysilylether), ester, carbonate, carbamates, sulfonate, andphosphate protecting groups. Examples of hydroxy protecting groupsinclude, but are not limited to, methyl ether; methoxymethyl ether,methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether,benzyloxymethyl ether, p-methoxybenzyloxymethyl ether,p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether,(4-methoxyphenoxy)methyl ether, guaiacolmethyl ether, t-butoxymethylether, 4-pentenyloxymethyl ether, siloxymethyl ether,2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether,bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether,menthoxymethyl ether, tetrahydropyranyl ether, 1-methoxycylcohexylether, 4-methoxytetrahydrothiopyranyl ether,4-methoxytetrahydrothiopyranyl ether S,S-Dioxide,1-R2-choro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether,1-(2-fluorophneyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl ether,tetrahydrofuranyl ether, tetrahydrothiofuranyl ether; substituted ethylethers such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether,1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether,1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethylether, 1-methyl-1phenoxyethyl ether, 2-trimethylsilyl ether, t-butylether, allyl ether, propargyl ethers, p-chlorophenyl ether,p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,tripropylsilylether, dimethylisopropylsilyl ether, diethylisopropylsilylether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether,diphenylmethylsilyl ether, benzoylformate ester, acetate ester,chloroacetate ester, dichloroacetate ester, trichloroacetate ester,trifluoroacetate ester, methoxyacetate ester, triphneylmethoxyacetateester, phenylacetate ester, benzoate ester, alkyl methyl carbonate,alkyl 9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl2,2,2,-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethylcarbonate, alkylsulfonate, methanesulfonate, benzylsulfonate, tosylate,methylene acetal, ethylidene acetal, and t-butylmethylidene ketal.Preferred protecting groups are represented by the formulas —R^(a),—Si(R^(a))(R^(a))(R^(a)), —C(O)R^(a), —C(O)OR^(a), —C(O)NH(R^(a)),—S(O)₂R^(a), —S(O)₂OH, P(O)(OH)₂, and —P(O)(OH)OR^(a), wherein R^(a) isC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —C₁-C₂₀alkylene(carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), —C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle) wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicalswhether alone or as part of another group are optionally substituted.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 161P2F10B (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 161P2F10B. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g.,a 161P2F10B-related protein). For example, an analog of a 161P2F10Bprotein can be specifically bound by an antibody or T cell thatspecifically binds to 161P2F10B.

The term “antibody” is used in the broadest sense unless clearlyindicated otherwise. Therefore, an “antibody” can be naturally occurringor man-made such as monoclonal antibodies produced by conventionalhybridoma technology. 161P2F10B antibodies comprise monoclonal andpolyclonal antibodies as well as fragments containing theantigen-binding domain and/or one or more complementarity determiningregions of these antibodies. As used herein, the term “antibody” refersto any form of antibody or fragment thereof that specifically binds161P2F10B and/or exhibits the desired biological activity andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyspecifically bind 161P2F10B and/or exhibit the desired biologicalactivity. Any specific antibody can be used in the methods andcompositions provided herein. Thus, in one embodiment the term“antibody” encompasses a molecule comprising at least one variableregion from a light chain immunoglobulin molecule and at least onevariable region from a heavy chain molecule that in combination form aspecific binding site for the target antigen. In one embodiment, theantibody is an IgG antibody. For example, the antibody is a IgG1, IgG2,IgG3, or IgG4 antibody. The antibodies useful in the present methods andcompositions can be generated in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes.Therefore, in one embodiment, an antibody of the present invention is amammalian antibody. Phage techniques can be used to isolate an initialantibody or to generate variants with altered specificity or aviditycharacteristics. Such techniques are routine and well known in the art.In one embodiment, the antibody is produced by recombinant means knownin the art. For example, a recombinant antibody can be produced bytransfecting a host cell with a vector comprising a DNA sequenceencoding the antibody. One or more vectors can be used to transfect theDNA sequence expressing at least one VL and one VH region in the hostcell. Exemplary descriptions of recombinant means of antibody generationand production include Delves, Antibody Production: Essential Techniques(Wiley, 1997); Shephard, et al., Monoclonal Antibodies (OxfordUniversity Press, 2000); Goding, Monoclonal Antibodies: Principles AndPractice (Academic Press, 1993); Current Protocols In Immunology (JohnWiley & Sons, most recent edition). An antibody of the present inventioncan be modified by recombinant means to increase efficacy of theantibody in mediating the desired function. Thus, it is within the scopeof the invention that antibodies can be modified by substitutions usingrecombinant means. Typically, the substitutions will be conservativesubstitutions. For example, at least one amino acid in the constantregion of the antibody can be replaced with a different residue. See,e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No.WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). Themodification in amino acids includes deletions, additions, andsubstitutions of amino acids. In some cases, such changes are made toreduce undesired activities, e.g., complement-dependent cytotoxicity.Frequently, the antibodies are labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature. Theseantibodies can be screened for binding to normal or defective 161P2F10B.See e.g., Antibody Engineering: A Practical Approach (Oxford UniversityPress, 1996). Suitable antibodies with the desired biologic activitiescan be identified the following in vitro assays including but notlimited to: proliferation, migration, adhesion, soft agar growth,angiogenesis, cell-cell communication, apoptosis, transport, signaltransduction, liternalization, antibody mediated secondary killing, andthe following in vivo assays such as the inhibition of tumor growth. Theantibody provided herein can be useful as an intermediate of ADC. Theantibodies provided herein can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for the ability to bind to the specific antigen withoutinhibiting the receptor-binding or biological activity of the antigen.As neutralizing antibodies, the antibodies can be useful in competitivebinding assays. They can also be used to quantify the 161P2F10B or itsreceptor.

The term “antigen-binding portion” or “antibody fragment” of an antibody(or simply “antibody portion”), as used herein, refers to one or morefragments of a 161P2F10B antibody that retain the ability tospecifically bind to an antigen (e.g., 161P2F10B; FIG. 1). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) an Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), C_(L) and C_(H1) domains; (ii) an F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) an Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) an Fv fragment consisting of the V_(L) andV_(H) domains of a single arm of an antibody, (v) a dAb fragment (Ward,et al. (1989) Nature 341:544-546), which consists of a V_(H) domain; and(vi) an isolated complementarily determining region (CDR). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see, e.g., Bird, et al.(1988) Science 242:423-426; and Huston, et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883). Such single chain antibodies are also intendedto be encompassed within the term “antigen-binding portion” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

As used herein, any form of the “antigen” can be used to generate anantibody that is specific for 161P2F10B. Thus, the eliciting antigen maybe a single epitope, multiple epitopes, or the entire protein alone orin combination with one or more immunogenicity enhancing agents known inthe art. The eliciting antigen may be an isolated full-length protein, acell surface protein (e g , immunizing with cells transfected with atleast a portion of the antigen), or a soluble protein (e.g., immunizingwith only the extracellular domain portion of the protein). The antigenmay be produced in a genetically modified cell. The DNA encoding theantigen may be genomic or non-genomic (e.g., cDNA) and encodes at leasta portion of the extracellular domain. As used herein, the term“portion” refers to the minimal number of amino acids or nucleic acids,as appropriate, to constitute an immunogenic epitope of the antigen ofinterest. Any genetic vectors suitable for transformation of the cellsof interest may be employed, including but not limited to adenoviralvectors, plasmids, and non-viral vectors, such as cationic lipids. Inone embodiment, the antibody of the methods and compositions hereinspecifically bind at least a portion of the extracellular domain of the161P2F10B of interest.

The antibodies or antigen binding fragments thereof provided herein maybe conjugated to a “bioactive agent.” As used herein, the term“bioactive agent” refers to any synthetic or naturally occurringcompound that binds the antigen and/or enhances or mediates a desiredbiological effect to enhance cell-killing toxins. In one embodiment, thebinding fragments useful in the present invention are biologicallyactive fragments. As used herein, the term “biologically active” refersto an antibody or antibody fragment that is capable of binding thedesired antigenic epitope and directly or indirectly exerting a biologiceffect. Direct effects include, but are not limited to the modulation,stimulation, and/or inhibition of a growth signal, the modulation,stimulation, and/or inhibition of an anti-apoptotic signal, themodulation, stimulation, and/or inhibition of an apoptotic or necroticsignal, modulation, stimulation, and/or inhibition the ADCC cascade, andmodulation, stimulation, and/or inhibition the CDC cascade.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they specifically bindthe target antigen and/or exhibit the desired biological activity (U.S.Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “Chemotherapeutic Agent” refers to all chemical compounds thatare effective in inhibiting tumor growth. Non-limiting examples ofchemotherapeutic agents include alkylating agents; for example, nitrogenmustards, ethyleneimine compounds and alkyl sulphonates;antimetabolites, for example, folic acid, purine or pyrimidineantagonists; mitotic inhibitors, for example, anti-tubulin agents suchas vinca alkaloids, auristatins and derivatives of podophyllotoxin;cytotoxic antibiotics; compounds that damage or interfere with DNAexpression or replication, for example, DNA minor groove binders; andgrowth factor receptor antagonists. In addition, chemotherapeutic agentsinclude cytotoxic agents (as defined herein), antibodies, biologicalmolecules and small molecules.

The term “compound” refers to and encompasses the chemical compounditself as well as, whether explicitly stated or not, and unless thecontext makes clear that the following are to be excluded: amorphous andcrystalline forms of the compound, including polymorphic forms, wherethese forms may be part of a mixture or in isolation; free acid and freebase forms of the compound, which are typically the forms shown in thestructures provided herein; isomers of the compound, which refers tooptical isomers, and tautomeric isomers, where optical isomers includeenantiomers and diastereomers, chiral isomers and non-chiral isomers,and the optical isomers include isolated optical isomers as well asmixtures of optical isomers including racemic and non-racemic mixtures;where an isomer may be in isolated form or in a mixture with one or moreother isomers; isotopes of the compound, including deuterium- andtritium-containing compounds, and including compounds containingradioisotopes, including therapeutically- and diagnostically-effectiveradioisotopes; multimeric forms of the compound, including dimeric,trimeric, etc., forms; salts of the compound, preferablypharmaceutically acceptable salts, including acid addition salts andbase addition salts, including salts having organic counterions andinorganic counterions, and including zwitterionic forms, where if acompound is associated with two or more counterions, the two or morecounterions may be the same or different; and solvates of the compound,including hemisolvates, monosolvates, disolvates, etc., includingorganic solvates and inorganic solvates, said inorganic solvatesincluding hydrates; where if a compound is associated with two or moresolvent molecules, the two or more solvent molecules may be the same ordifferent. In some instances, reference made herein to a compound of theinvention will include an explicit reference to one or of the aboveforms, e.g., salts and/or solvates, however, this reference is foremphasis only, and is not to be construed as excluding other of theabove forms as identified above.

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes, chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins (e.g.,auristatin e, auristatin f, MMAE and MMAF), auromycins, maytansinoids,ricin, ricin A-chain, combretastatin, duocarmycins, dolastatins,doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹²or ²¹³ , P³² and radioactive isotopes of Lu including Lu¹⁷⁷. Antibodiesmay also be conjugated to an anti-cancer pro-drug activating enzymecapable of converting the pro-drug to its active form.

The term “deplete,” in the context of the effect of a 161P2F10B bindingagent on 161P2F10B-expressing cells, refers to a reduction in the numberof or elimination of the 161P2F10B-expressing cells.

The “gene product” is used herein to indicate a peptide/protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 1. The cancer protein can be a fragment, oralternatively, be the full-length protein encoded by nucleic acids ofFIG. 1. In one embodiment, a cancer amino acid sequence is used todetermine sequence identity or similarity. In another embodiment, thesequences are naturally occurring allelic variants of a protein encodedby a nucleic acid of FIG. 1. In another embodiment, the sequences aresequence variants as further described herein.

“Heteroconjugate” antibodies are useful in the present methods andcompositions. As used herein, the term “heteroconjugate antibody” refersto two covalently joined antibodies. Such antibodies can be preparedusing known methods in synthetic protein chemistry, including usingcrosslinking agents. See, e.g., U.S. Pat. No. 4,676,980.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a “human antibody.”As used herein, the term “human antibody” refers to an antibody in whichessentially the entire sequences of the light chain and heavy chainsequences, including the complementary determining regions (CDRs), arefrom human genes. In one embodiment, human monoclonal antibodies areprepared by the trioma technique, the human B-cell technique (see, e.g.,Kozbor, et al., Immunol. Today 4: 72 (1983), EBV transformationtechnique (see, e.g., Cole, et al. Monoclonal Antibodies And CancerTherapy 77-96 (1985)), or using phage display (see, e.g., Marks, et al.,J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the humanantibody is generated in a transgenic mouse. Techniques for making suchpartially to fully human antibodies are known in the art and any suchtechniques can be used. According to one particularly preferredembodiment, fully human antibody sequences are made in a transgenicmouse engineered to express human heavy and light chain antibody genes.An exemplary description of preparing transgenic mice that produce humanantibodies found in Application No. WO02/43478 and U.S. Pat. No.6,657,103 (Abgenix) and its progeny. B cells from transgenic mice thatproduce the desired antibody can then be fused to make hybridoma celllines for continuous production of the antibody. See, e.g., U.S. Pat.Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; andJakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp.Med. 188:483-95 (1998).

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

Suitable “labels” include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. In addition, the antibodies provided hereincan be useful as the antigen-binding component of fluorobodies. Seee.g., Zeytun, et al., Nat. Biotechnol. 21:1473-79 (2003).

The terms “metastatic cancer” and “metastatic disease” mean cancers thathave spread to regional lymph nodes or to distant sites, and are meantto include stage D disease under the AUA system and stage T×N×M+ underthe TNM system.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g., to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates, or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. In oneembodiment, the polyclonal antibody contains a plurality of monoclonalantibodies with different epitope specificities, affinities, oravidities within a single antigen that contains multiple antigenicepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler, et al., Nature256: 495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson, et al., Nature 352: 624-628 (1991) and Marks, et al., J. Mol.Biol. 222: 581-597 (1991), for example. These monoclonal antibodies willusually bind with at least a Kd of about 1 μM, more usually at leastabout 300 nM, typically at least about 30 nM, preferably at least about10 nM, more preferably at least about 3 nM or better, usually determinedby ELISA.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 1, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

As used herein, the terms “specific”, “specifically binds” and “bindsspecifically” refer to the selective binding of the antibody to thetarget antigen epitope. Antibodies can be tested for specificity ofbinding by comparing binding to appropriate antigen to binding toirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to the appropriate antigen at least 2, 5, 7, andpreferably 10 times more than to irrelevant antigen or antigen mixturethen it is considered to be specific. In one embodiment, a specificantibody is one that binds the 161P2F10B-related antigen (particularly161P2F10B), but does not bind to the irrelevant antigen.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; as isreadily appreciated in the art, full eradication of disease is apreferred out albeit not a requirement for a treatment act.

The “161P2F10B-related proteins” include those specifically identifiedherein (see, FIG. 1), as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 161P2F10B proteins orfragments thereof, as well as fusion proteins of a 161P2F10B protein anda heterologous polypeptide are also included. Such 161P2F10B proteinsare collectively referred to as the 161P2F10B-related proteins, theproteins of the invention, or 161P2F10B. The term “161P2F10B-relatedprotein” refers to a polypeptide fragment or a 161P2F10B proteinsequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30,35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids.

II.) 161P2F10B Antibodies

Antibodies for ADCs of the invention specifically bind to a 161P2F10Bprotein and do not bind (or bind weakly) to peptides or proteins thatare not 161P2F10B-related proteins under physiological conditions.

Various methods for the preparation of antibodies, specificallymonoclonal antibodies, are well known in the art. For example,antibodies can be prepared by immunizing a suitable mammalian host usinga 161P2F10B-related protein, peptide, or fragment thereof, in isolatedor immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press,Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring HarborPress, NY (1989)). In addition, fusion proteins of 161P2F10B can also beused, such as a 161P2F10B GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 1 is produced, and then used as an immunogen togenerate appropriate antibodies. In another embodiment, a161P2F10B-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 161P2F10B-related protein or 161P2F10Bexpressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly, et al., 1997, Ann. Rev. Immunol.15: 617-648).

The amino acid sequence of a 161P2F10B protein as shown in FIG. 1 can beanalyzed to select specific regions of the 161P2F10B protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 161P2F10B amino acid sequence are used to identifyhydrophilic regions in the 161P2F10B structure. Regions of a 161P2F10Bprotein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Preferred methods for the generation of161P2F10B antibodies are further illustrated by way of the examplesprovided herein. Methods for preparing a protein or polypeptide for useas an immunogen are well known in the art. Also well known in the artare methods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 161P2F10B immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

In a preferred embodiment, a 161P2F10B MAb for ADCs of the inventioncomprises heavy and light chain variable regions of an antibodydesignated H16-7.8 (See, FIG. 3), or heavy and light variable regionscomprising amino acid sequences that are homologous to the amino acidsequences of the heavy and light chain variable regions of H16-7.8, andwherein the antibodies retain the desired functional properties(particularly binding activity to 161P2F10B) of the 161P2F10B MAbs ofthe invention. The heavy chain variable region of H16-7.8 consists ofthe amino acid sequence ranging from 20^(th) Q residue to the 142^(nd) Sresidue of SEQ ID NO:7, and the light chain variable region of H16-7.8consists of the amino acid sequence ranging from 20^(th) E residue tothe 127^(th) R residue of SEQ ID NO:8. As the constant region of theantibody of the invention, any subclass of constant region can bechosen. Preferably, human IgG2 constant region as the heavy chainconstant region and human Ig kappa constant region as the light chainconstant region can be used. Reactivity (binding activity) of 161P2F10BMAbs with a 161P2F10B protein can be established by a number of wellknown means, including Western blot, immunoprecipitation, ELISA, andFACS analyses using161P2F10B proteins, 161P2F10B-expressing cells orextracts thereof.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer, et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe 161P2F10B MAb.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the 161P2F10B MAb. Morespecifically, one or more amino acid mutations are introduced into theCH2-CH3 domain interface region of the Fc-hinge fragment such that theantibody has impaired Staphylococcal protein A (SpA) binding relative tonative Fc-hinge domain SpA binding. This approach is described infurther detail in U.S. Pat. No. 6,165,745 by Ward, et al.

In another embodiment, the 161P2F10B MAb is modified to increase itsbiological half life. Various approaches are possible. For example,mutations can be introduced as described in U.S. Pat. No. 6,277,375 toWard. Alternatively, to increase the biological half life, the antibodycan be altered within the CH1 or CL region to contain a salvage receptorbinding epitope taken from two loops of a CH2 domain of an Fc region ofan IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 byPresta, et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the 161P2F10B MAb. For example, one or moreamino acids selected from amino acid specific residues can be replacedwith a different amino acid residue such that the antibody has analtered affinity for an effector ligand but retains the antigen-bindingability of the parent antibody. The effector ligand to which affinity isaltered can be, for example, an Fc receptor or the C1 component ofcomplement. This approach is described in further detail in U.S. Pat.Nos. 5,624,821 and 5,648,260, both by Winter, et al.

In one embodiment, the antibody for ADC of the present invention isproduced by recombinant means known in the art. For example, arecombinant antibody can be produced by transfecting a host cell with avector comprising a DNA sequence encoding the antibody. One or morevectors can be used to transfect the DNA sequence expressing at leastone VL and one VH region in the host cell. Exemplary descriptions ofrecombinant means of antibody generation and production include Delves,ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, etal., Monoclonal Antibodies (Oxford University Press, 2000); Goding,Monoclonal Antibodies: Principles And Practice (Academic Press, 1993);Current Protocols In Immunology (John Wiley & Sons, most recentedition).

The antibody for ADC of the present invention can easily be prepared bythose skilled in the art on the basis of the sequence information on theheavy-chain variable region and light-chain variable region thereofdisclosed herein, using a method commonly known in the art.Specifically, a heavy-chain variable region gene fragment having a basesequence that encodes the heavy-chain variable region amino acid of theantibody for ADC of the present invention (SEQ ID NO:7, from 20 to 142),and a light-chain variable region gene fragment having a base sequencethat encodes the light-chain variable region amino acid of the antibodyfor ADC of the present invention (SEQ ID NO:8, from 20 to 127) areprepared. Then, the variable region genes are joined to a constantregion gene in an appropriate class of human antibody to prepare anantibody gene. Next, this antibody gene is joined to an appropriateexpression vector, and introduced to a cultured cell. Finally, thiscultured cell is cultured, whereby the antibody can be obtained from theculture supernatant.

Each of the above-described variable region gene fragments that encodethe heavy-chain and light-chain variable region amino acids of theantibody of the present invention (SEQ ID NO:7, from 20 to 142 and SEQID NO:8, from 20 to 127) can be prepared by on the basis of basesequences designed on the basis of the amino acid sequences of theheavy-chain and light-chain variable regions (SEQ ID NO:7, from 20 to142 and SEQ ID NO:8, from 20 to 127), or on the basis of the basesequences of the heavy-chain and light-chain variable regions of theantibody of the present invention, shown by SEQ ID NO:4, from 91 to 459and SEQ ID NO:6, from 100 to 423, using a method of gene synthesiscommonly known in the art. As such a method of gene synthesis, variousmethods obvious to those skilled in the art, such as the antibody genesynthesis method described in WO90/07861, can be used. Next, theabove-described variable region gene fragments and the constant regiongene of the human antibody are joined to prepare an antibody gene.Although any subclass of constant region can be chosen as the constantregion of the human antibody used, human Ig[gamma]2 as the heavy-chainconstant region, and human Ig[kappa] as the light-chain constant region,can be preferably used.

As the preferable antibody heavy-chain gene of the antibody for ADC ofpresent invention, obtained by joining the heavy-chain variable regiongene shown by SEQ ID NO:7, from 20 to 142 and the human Ig[gamma]2heavy-chain constant region gene, a gene comprising a base sequence thatencodes the amino acid sequence shown by SEQ ID NO:7, from 20 to 468,more preferably a gene comprising the base sequence shown by SEQ IDNO:4, from 91 to 1437, can be mentioned. As the preferable antibodylight-chain gene of the antibody for ADC of present invention, obtainedby joining the light-chain variable region gene shown by SEQ ID NO:8,from 20 to 127 and the human Ig[kappa] light-chain constant region gene,a gene comprising a base sequence that encodes the amino acid sequenceshown by SEQ ID NO:8, from 20 to 233, more preferably a gene comprisingthe base sequence shown by SEQ ID NO:6, from 100 to 741, can bementioned. As the antibody for ADC of the present invention, encoded bya heavy-chain gene comprising the base sequence shown by SEQ ID NO:4,from 91 to 1437 and a light-chain gene comprising the base sequenceshown by SEQ ID NO:6, from 100 to 741, H16-7.8, described in an Examplebelow, can be mentioned.

Subsequent to the preparation of this antibody gene, introduction of theantibody gene to an expression vector, introduction of the expressionvector to cultured cells, cultivation of the cultured cells,purification of the antibody and the like can be performed by usingvarious methods commonly known in the art. The expression vector is notsubject to limitation, as long as it is capable of expressing theantibody gene. It is preferable to utilize an expression vector alreadyhaving a human Ig constant region gene such as AG-[gamma]2 orAG-[kappa], because it would become an expression vector having theantibody gene simply when the antibody variable region gene is insertedthereto.

The above-described expression vector is introduced to cultured cellsby, for example, the calcium phosphate method and the like. As examplesof the cultured cells to which the expression vector is introduced,cultured cells such as CHO cells can be used, and they may be culturedby a conventional method. After the above-described cultivation, theantibody accumulated in the culture supernatant can be purified by, forexample, various chromatographies using a Protein A column.

Reactivity(binding activity) of 161P2F10B antibodies thus obtained witha 161P2F10B protein can be established by a number of well known means,including Western blot, immunoprecipitation, ELISA, and FACS analysesusing, as appropriate, 161P2F10B protein, 161P2F10B-expressing cells orextracts thereof. The antibody thus obtained or an antibody fragmentretaining an activity due to the antibody after being further purifiedas required, can be prepared as an antibody for ADC. A 161P2F10Bantibody or fragment thereof can be labeled with a detectable marker orconjugated to a second molecule. Suitable detectable markers include,but are not limited to, a radioisotope, a fluorescent compound, abioluminescent compound, chemiluminescent compound, a metal chelator oran enzyme. Further, bi-specific antibodies specific for two or more161P2F10B epitopes are generated using methods generally known in theart. Homodimeric antibodies can also be generated by cross-linkingtechniques known in the art (e.g., Wolff, et al., Cancer Res. 53:2560-2565).

In yet another preferred embodiment, the 161P2F10B MAb for ADCs of theinvention is an antibody comprising heavy and light chain of an antibodydesignated H16-7.8. The heavy chain of H16-7.8 consists of the aminoacid sequence ranging from 20^(th) Q residue to the 468^(th) K residueof SEQ ID NO:7 and the light chain of H16-7.8 consists of amino acidsequence ranging from 20^(th) E residue to the 233^(th) C residue of SEQID NO:8 sequence. The sequence of which is set forth in FIG. 2 and FIG.3. In a preferred embodiment, H16-7.8 is conjugated to a cytotoxicagent.

III.) Antibody-Drug Conjugates Generally

In another aspect, the invention provides antibody-drug conjugates(ADCs), comprising an antibody conjugated to a cytotoxic agent such asMMAF. In another aspect, the invention further provides methods of usingthe ADCs. In one aspect, an ADC comprises any of the above 161P2F10BMAbs covalently attached to a cytotoxic agent.

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin, et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera, et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland, et al. (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland, et al., (1986)supra). Toxins used in antibody-toxin conjugates include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler, et al. (2000) Jour. ofthe Nat. Cancer Inst. 92(19):1573-1581; Mandler, et al. (2000)Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler, et al. (2002)Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu, et al.(1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin(Lode, et al. (1998) Cancer Res. 58:2928; Hinman, et al. (1993) CancerRes. 53:3336-3342). The toxins may effect their cytotoxic and cytostaticeffects by mechanisms including tubulin binding, DNA binding, ortopoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

Examples of antibody drug conjugates are, ZEVALIN® (ibritumomabtiuxetan, Biogen/Idec) which is an antibody-radioisotope conjugatecomposed of a murine IgG1 kappa monoclonal antibody directed against theCD20 antigen found on the surface of normal and malignant B lymphocytesand ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourea linker-chelator(Wiseman, et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman, etal. (2002) Blood 99(12):4336-42; Witzig, et al. (2002) J. Clin. Oncol.20(10):2453-63; Witzig, et al. (2002) J. Clin. Oncol. 20(15):3262-69).

Additionally, MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals),an antibody drug conjugate composed of a hu CD33 antibody linked tocalicheamicin, was approved in 2000 for the treatment of acute myeloidleukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat.Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;5,767,285; and 5,773,001).

In addition, Cantuzumab mertansine (Immunogen, Inc.), an antibody drugconjugate composed of the huC242 antibody linked via the disulfidelinker SPP to the maytansinoid drug moiety, DM1, is advancing into PhaseII trials for the treatment of cancers that express CanAg, such ascolon, pancreatic, gastric, and others.

Additionally, MLN-2704 (Millennium Pharm., BZL Biologics, ImmunogenInc.), an antibody drug conjugate composed of the anti-prostate specificmembrane antigen (PSMA) monoclonal antibody linked to the maytansinoiddrug moiety, DM1, is under development for the potential treatment ofprostate tumors.

Finally, the auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronin, et al. (2003) Nature Biotechnology21(7):778-784) and are under therapeutic development.

III (A). Auristatins and Dolostatins

Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke, et al. (2001) Antimicrob. Agents and Chemother.45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) andantifungal activity (Pettit, et al. (1998) Antimicrob. Agents Chemother.42:2961-2965). The dolastatin or auristatin drug moiety may be attachedto the antibody through the N (amino) terminus or the C (carboxyl)terminus of the peptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in Senter, etal, “Proceedings of the American Association for Cancer Research,”Volume 45, Abstract Number 623, and presented Mar. 28, 2004, thedisclosure of which is expressly incorporated by reference in itsentirety.

An exemplary auristatin embodiment is MMAE (wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate).

Another exemplary auristatin embodiment is MMAF, wherein the wavy lineindicates the covalent attachment to a linker (L) of an antibody drugconjugate (US 2005/0238649):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 12):

ADC of the present invention include MMAF. The preferred embodiment, theADC of the present invention include maleimidocaproyl (mc) as a linkerand MMAF as a drug.

IV.) Antibody-Drug Conjugate Compounds which Bind 161P2F10B

The present invention provides, inter alia, antibody-drug conjugatecompounds for targeted delivery of drugs. The inventors have made thediscovery that the antibody-drug conjugate compounds have potentcytotoxic and/or cytostatic activity against cells expressing 161P2F10B.The antibody-drug conjugate compounds comprise an Antibody unitcovalently linked to at least one Drug unit. The Drug units can becovalently linked directly or via a Linker unit (-LU-).

In some embodiments, the antibody drug conjugate compound has thefollowing formula:

L-(LU-D)_(p)   (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein:

L is the Antibody unit, e.g., 161P2F10B MAb of the present invention,and

(LU-D) is a Linker unit-Drug unit moiety, wherein:

LU- is a Linker unit, and

-D is a drug unit having cytostatic or cytotoxic activity against atarget cell; and

p is an integer from 1 to 20.

In some embodiments, p ranges from 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, pranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2to 3. In other embodiments, p is 1, 2, 3, 4, 5 or 6. In someembodiments, p is 2 or 4.

In some embodiments, the antibody drug conjugate compound has thefollowing formula:

L-(A_(a)-W_(w)-Y_(y)-D)_(p)   (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

L is the Antibody unit, e.g., 161P2F10B MAb; and

-A_(a)-W_(w)-Y_(y)- is a Linker unit (LU), wherein:

-A- is a Stretcher unit,

a is 0 or 1,

each -W- is independently an Amino Acid unit,

w is an integer ranging from 0 to 12,

-Y- is a self-immolative spacer unit,

y is 0, 1 or 2;

-D is a drug units having cytostatic or cytotoxic activity against thetarget cell; and

p is an integer from 1 to 20.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In someembodiments, p ranges from 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p rangesfrom 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In otherembodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p is 2 or 4.In some embodiments, when w is not zero, y is 1 or 2. In someembodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 12 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined In someinstances, separation, purification, and characterization of homogeneousAntibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is from 2 to 8.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds comprise 161P2F10B MAb as the Antibodyunit, a drug, and optionally a linker that joins the drug and thebinding agent. In a preferred embodiment, the Antibody is 161P2F10B MAbcomprising heavy and light chain variable regions of an antibodydesignated H16-7.8 described above. In more preferred embodiment, theAntibody is 161P2F10B MAb comprising heavy and light chain of anantibody designated H16-7.8 described above. A number of differentreactions are available for covalent attachment of drugs and/or linkersto binding agents. This is often accomplished by reaction of the aminoacid residues of the binding agent, e.g., antibody molecule, includingthe amine groups of lysine, the free carboxylic acid groups of glutamicand aspartic acid, the sulfhydryl groups of cysteine and the variousmoieties of the aromatic amino acids. One of the most commonly usednon-specific methods of covalent attachment is the carbodiimide reactionto link a carboxy (or amino) group of a compound to amino (or carboxy)groups of the antibody. Additionally, bifunctional agents such asdialdehydes or imidoesters have been used to link the amino group of acompound to amino groups of an antibody molecule. Also available forattachment of drugs to binding agents is the Schiff base reaction. Thismethod involves the periodate oxidation of a drug that contains glycolor hydroxy groups, thus forming an aldehyde which is then reacted withthe binding agent. Attachment occurs via formation of a Schiff base withamino groups of the binding agent. Isothiocyanates can also be used ascoupling agents for covalently attaching drugs to binding agents. Othertechniques are known to the skilled artisan and within the scope of thepresent invention.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with the161P2F10B MAb under appropriate conditions.

Each of the particular units of the Antibody-drug conjugate compounds isdescribed in more detail herein. The synthesis and structure ofexemplary linker units, stretcher units, amino acid units,self-immolative spacer unit, and drug units are also described in U.S.Patent Application Publication Nos. 2003-0083263, 2005-0238649 and2005-0009751, each if which is incorporated herein by reference in itsentirety and for all purposes.

V.) Linker Units

Typically, the antibody-drug conjugate compounds comprise a linkerregion between the drug unit and the antibody unit. In some embodiments,the linker is cleavable under intracellular conditions, such thatcleavage of the linker releases the drug unit from the antibody in theintracellular environment. In yet other embodiments, the linker unit isnot cleavable and the drug is released, for example, by antibodydegradation.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (e.g., within a lysosome orendosome or caveolea). The linker can be, e.g., a peptidyl linker thatis cleaved by an intracellular peptidase or protease enzyme, including,but not limited to, a lysosomal or endosomal protease. In someembodiments, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in 161P2F10B-expressing cells. For example, a peptidyl linkerthat is cleavable by the thiol-dependent protease cathepsin-B, which ishighly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or aGly-Phe-Leu-Gly linker (SEQ ID NO:9). Other examples of such linkers aredescribed, e.g., in U.S. Pat. No. 6,214,345, incorporated herein byreference in its entirety and for all purposes. In a specificembodiment, the peptidyl linker cleavable by an intracellular proteaseis a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No.6,214,345, which describes the synthesis of doxorubicin with the val-citlinker). One advantage of using intracellular proteolytic release of thetherapeutic agent is that the agent is typically attenuated whenconjugated and the serum stabilities of the conjugates are typicallyhigh.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville, et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT (See, e.g., Thorpe, et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak, et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson, et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau, et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau, et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes). In preferred embodiment, linker unit is maleimidocaproyl(mc).

Typically, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (e.g., in plasma). Whether a linker is not substantiallysensitive to the extracellular environment can be determined, forexample, by incubating with plasma the antibody-drug conjugate compoundfor a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) andthen quantitating the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (i.e.,in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the 161P2F10B MAb.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO2004-010957, U.S.Publication No. 20060074008, U.S. Publication No. 20050238649, and U.S.Publication No. 20060024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

A “Linker unit” (LU) is a bifunctional compound that can be used to linka Drug unit and a Antibody unit to form an antibody-drug conjugatecompound. In some embodiments, the Linker unit has the formula:

-A_(a)-W_(w)-Y_(y)-

wherein: -A- is a Stretcher unit,

a is 0 or 1,

each -W- is independently an Amino Acid unit,

w is an integer ranging from 0 to 12,

-Y- is a self-immolative Spacer unit, and

y is 0, 1 or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In someembodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

VI.) The Stretcher Unit

The Stretcher unit (A), when present, is capable of linking an Antibodyunit to an Amino Acid unit (-W-), if present, to a Spacer unit (-Y-), ifpresent; or to a Drug unit (-D). Useful functional groups that can bepresent on a 161P2F10B MAb (e.g., H16-7.8), either naturally or viachemical manipulation include, but are not limited to, sulfhydryl,amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, andcarboxyl. Suitable functional groups are sulfhydryl and amino. In oneexample, sulfhydryl groups can be generated by reduction of theintramolecular disulfide bonds of a 161P2F10B MAb. In anotherembodiment, sulfhydryl groups can be generated by reaction of an aminogroup of a lysine moiety of a 161P2F10B MAb with 2-iminothiolane(Traut's reagent) or other sulfhydryl generating reagents. In certainembodiments, the 161P2F10B MAb is a recombinant antibody and isengineered to carry one or more lysines. In certain other embodiments,the recombinant 161P2F10B MAb is engineered to carry additionalsulfhydryl groups, e.g., additional cysteines.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Antibody unit. The sulfur atom can be derived from a sulfhydrylgroup of an antibody. Representative Stretcher units of this embodimentare depicted within the square brackets of Formulas IIIa and IIIb,wherein L-, -W-, -Y-, -D, w and y are as defined above, and R¹⁷ isselected from —C₁-C₁₀ alkylene-, —C₁-C₁₀ alkenylene-, —C₁-C₁₀alkynylene-, -carbocyclo-, —O—(C₁-C₈ alkylene)-, O—(C₁-C₈ alkenylene)-,—O—(C₁-C₈ alkynylene)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, —C₂-C₁₀alkenylene-arylene, —C₂-C₁₀ alkynylene-arylene, -arylene-C₁-C₁₀alkylene-, -arylene-C₂-C₁₀ alkenylene-, -arylene-C₂-C₁₀ alkynylene-,—C₁-C₁₀ alkylene-(carbocyclo)-, —C₂-C₁₀ alkenylene-(carbocyclo)-,—C₂-C₁₀ alkynylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-,-(carbocyclo)-C₂-C₁₀ alkenylene-, -(carbocyclo)-C₂-C₁₀ alkynylene,-heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, —C₂-C₁₀alkenylene-(heterocyclo)-, —C₂-C₁₀ alkynylene-(heterocyclo)-,-(heterocyclo)-C₁-C₁₀ alkylene-, -(heterocyclo)-C₂-C₁₀ alkenylene-,-(heterocyclo)-C₁-C₁₀ alkynylene-, —(CH₂CH₂O)_(r)—, or—(CH₂CH₂O)_(r)—CH₂—, and r is an integer ranging from 1-10, wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocycle, carbocyclo, heterocyclo, and arylene radicals, whether aloneor as part of another group, are optionally substituted. In someembodiments, said alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkynyklene, aryl, carbocyle, carbocyclo, heterocyclo, and aryleneradicals, whether alone or as part of another group, are unsubstituted.In some embodiments, R¹⁷ is selected from —C₁-C₁₀ alkylene-,-carbocyclo-, —O—(C₁-C₈ alkylene)-, -arylene-, —C₁-C₁₀alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, -(heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—; and r is an integerranging from 1-10, wherein said alkylene groups are unsubstituted andthe remainder of the groups are optionally substituted.

It is to be understood from all the exemplary embodiments that evenwhere not denoted expressly, from 1 to 12 drug moieties can be linked toan Antibody (p=1-12).

An illustrative Stretcher unit is that of Formula Ma wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

An illustrative Stretcher unit is that of Formula Ma wherein R¹⁷ is-arylene- or arylene-C₁-C₁₀ alkylene-. In some embodiments, the arylgroup is an unsubstituted phenyl group.

Still another illustrative Stretcher unit is that of Formula IIIbwherein R¹⁷ is —(CH₂)₅—:

In certain embodiments, the Stretcher unit is linked to the Antibodyunit via a disulfide bond between a sulfur atom of the Antibody unit anda sulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, L-, -W-, -Y-, -D, w and y are as defined above.

It should be noted that throughout this application, the S moiety in theformula below refers to a sulfur atom of the Antibody unit, unlessotherwise indicated by context.

In yet other embodiments, the Stretcher contains a reactive site thatcan form a bond with a primary or secondary amino group of an Antibody.Examples of these reactive sites include, but are not limited to,activated esters such as succinimide esters, 4 nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb, wherein —R¹⁷—, L-, -W-, -Y-,-D, w and y are as defined above;

In some embodiments, the Stretcher contains a reactive site that isreactive to a modified carbohydrate's (—CHO) group that can be presenton an Antibody. For example, a carbohydrate can be mildly oxidized usinga reagent such as sodium periodate and the resulting (—CHO) unit of theoxidized carbohydrate can be condensed with a Stretcher that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko, et al., 1991,Bioconjugate Chem. 2:133-41. Representative Stretcher units of thisembodiment are depicted within the square brackets of Formulas VIa, VIb,and VIc, wherein —R¹⁷—, L-, -W-, -Y-, -D, w and y are as defined asabove.

VII.) The Amino Acid Unit

The Amino Acid unit (-W-), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug moiety if the Spacer unit is absent, and links the Antibodyunit to the Drug unit if the Stretcher unit and Spacer unit are absent.

W_(w)- can be, for example, a monopeptide, dipeptide, tripeptide,tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide,nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each -W-unit independently has the formula denoted below in the square brackets,and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

In some embodiments, the Amino Acid unit can be enzymatically cleaved byone or more enzymes, including a cancer or tumor-associated protease, toliberate the Drug unit (-D), which in one embodiment is protonated invivo upon release to provide a Drug (D).

In certain embodiments, the Amino Acid unit can comprise natural aminoacids. In other embodiments, the Amino Acid unit can comprisenon-natural amino acids. Illustrative Ww units are represented byformulas (VII)-(IX):

wherein R²⁰ and R²¹ are as follows:

R²⁰ R²¹ Benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R²⁰, R²¹ and R²² are as follows:

R²⁰ R²¹ R²² benzyl benzyl (CH₂)₄NH₂; isopropyl benzyl (CH₂)₄NH₂; and Hbenzyl (CH₂)₄NH₂;

wherein R²⁰, R²¹, R²² and R²³ are as follows:

R²⁰ R²¹ R²² R²³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.

Exemplary Amino Acid units include, but are not limited to, units offormula VII where: R²⁰ is benzyl and R²¹ is —(CH₂)₄NH₂; R²⁰ is isopropyland R²¹ is —(CH₂)₄NH₂; or R²⁰ is isopropyl and R²¹ is —(CH₂)₃NHCONH₂.Another exemplary Amino Acid unit is a unit of formula VIII wherein R²⁰is benzyl, R²¹ is benzyl, and R²² is —(CH₂)₄NH₂.

Useful -W_(w)- units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzyme, for example, atumor-associated protease. In one embodiment, a -W_(w)- unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In one embodiment, -W_(w)- is a dipeptide, tripeptide, tetrapeptide orpentapeptide. When R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, thecarbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached isindependently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit isvaline-citrulline (vc or val-cit). In another aspect, the Amino Acidunit is phenylalanine-lysine (i.e., fk). In yet another aspect of theAmino Acid unit, the Amino Acid unit is N-methylvaline-citrulline. Inyet another aspect, the Amino Acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinolinecarboxylate lysine,cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine,glycine serine valine glutamine and isonepecotic acid.

VIII.) The Spacer Unit

The Spacer unit (-Y-), when present, links an Amino Acid unit to theDrug unit when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug unit when the Amino Acid unitis absent. The Spacer unit also links the Drug unit to the Antibody unitwhen both the Amino Acid unit and Stretcher unit are absent.

Spacer units are of two general types: non self-immolative orself-immolative. A non self-immolative Spacer unit is one in which partor all of the Spacer unit remains bound to the Drug moiety aftercleavage, particularly enzymatic, of an Amino Acid unit from theantibody-drug conjugate. Examples of a non self-immolative Spacer unitinclude, but are not limited to a (glycine-glycine) Spacer unit and aglycine Spacer unit (both depicted in Scheme 1) (infra). When aconjugate containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via an enzyme (e.g., a tumor-cellassociated-protease, a cancer-cell-associated protease or alymphocyte-associated protease), a glycine-glycine-Drug moiety or aglycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, anindependent hydrolysis reaction takes place within the target cell,cleaving the glycine-Drug moiety bond and liberating the Drug.

In some embodiments, a non self-immolative Spacer unit (-Y-) is -Gly-.In some embodiments, a non self-immolative Spacer unit (-Y-) is-Gly-Gly-.

In one embodiment, a Drug-Linker conjugate is provided in which theSpacer unit is absent (y=0), or a pharmaceutically acceptable salt orsolvate thereof.

Alternatively, a conjugate containing a self-immolative Spacer unit canrelease -D. As used herein, the term “self-immolative Spacer” refers toa bifunctional chemical moiety that is capable of covalently linkingtogether two spaced chemical moieties into a stable tripartite molecule.It will spontaneously separate from the second chemical moiety if itsbond to the first moiety is cleaved.

In some embodiments, -Y_(y)- is a p-aminobenzyl alcohol (PAB) unit (seeSchemes 2 and 3) whose phenylene portion is substituted with Q_(m)wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4. The alkyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted.

In some embodiments, -Y- is a PAB group that is linked to -W_(w)- viathe amino nitrogen atom of the PAB group, and connected directly to -Dvia a carbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, Scheme 2 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via acarbamate or carbonate group as described by Toki et al., 2002, J. Org.Chem. 67:1866-1872.

In Scheme 2, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 12. The alkyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted.

Without being bound by any particular theory or mechanism, Scheme 3depicts a possible mechanism of Drug release of a PAB group which isattached directly to -D via an ether or amine linkage, wherein Dincludes the oxygen or nitrogen group that is part of the Drug unit.

In Scheme 3, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 12. The alkyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay, et al., 1999,Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues, et al., 1995, Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, etal., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry, et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples ofself-immolative spacers.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, whichcan be used to incorporate and release multiple drugs.

In Scheme 4, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and pranges raging from 1 to about 12. The alkyl, alkenyl and alkynyl groups,whether alone or as part of another group, can be optionallysubstituted.

In some embodiments, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4. The alkyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted.

Embodiments of the Formula I and II comprising antibody-drug conjugatecompounds can include:

wherein w and y are each 0, 1 or 2, and,

wherein w and y are each 0,

IX.) The Drug Unit

The Drug moiety (D) can be any cytotoxic, cytostatic or immunomodulatory(e.g., immunosuppressive) or drug. D is a Drug unit (moiety) having anatom that can form a bond with the Spacer unit, with the Amino Acidunit, with the Stretcher unit or with the Antibody unit. In someembodiments, the Drug unit D has a nitrogen atom that can form a bondwith the Spacer unit. As used herein, the terms “drug unit” and “drugmoiety” are synonymous and used interchangeably.

Useful classes of cytotoxic or immunomodulatory agents include, forexample, antitubulin agents, DNA minor groove binders, DNA replicationinhibitors, and alkylating agents.

In some embodiments, the Drug is an auristatin, such as auristatin E(also known in the art as a derivative of dolastatin-10) or a derivativethereof. The auristatin can be, for example, an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatins include AFP, MMAF, andMMAE. The synthesis and structure of exemplary auristatins are describedin U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649and 2005-0009751; International Patent Publication No. WO04/010957,International Patent Publication No. WO02/088172, and U.S. Pat. Nos.6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;4,816,444; and 4,486,414, each of which is incorporated by referenceherein in its entirety and for all purposes.

Auristatins have been shown to interfere with microtubule dynamics andnuclear and cellular division and have anticancer activity. MMAF bindtubulin and can exert a cytotoxic or cytostatic effect on a161P2F10B-expressing cell. There are a number of different assays, knownin the art, which can be used for determining whether an auristatin orresultant antibody-drug conjugate exerts a cytostatic or cytotoxiceffect on a desired cell line.

Methods for determining whether a compound binds tubulin are known inthe art. See, for example, Muller, et al., Anal. Chem 2006, 78,4390-4397; Hamel, et al., Molecular Pharmacology, 1995 47: 965-976; andHamel, et al., The Journal of Biological Chemistry, 1990 265:28,17141-17149. For purposes of the present invention, the relativeaffinity of a compound to tubulin can be determined Some preferredauristatins of the present invention bind tubulin with an affinityranging from 10 fold lower (weaker affinity) than the binding affinityof MMAE to tubulin to 10 fold, 20 fold or even 100 fold higher (higheraffinity) than the binding affinity of MMAE to tublin.

In some embodiments, -D is an auristatin of the formula D_(E) or D_(F):

or a pharmaceutically acceptable salt or solvate form thereof;

wherein, independently at each location:

the wavy line indicates a bond;

R² is —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R³ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), -aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alyklene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), -heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁵ is —H or —C₁-C₈ alkyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(s)— wherein R^(a) and R^(b) are independently —H,—C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, or -carbocycle and s is2, 3, 4, 5 or 6,

R⁶ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R⁷ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), -aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

each R⁸ is independently —H, —OH, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl,—C₂-C₂₀ alkynyl, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₁-C₂₀alkynyl), or -carbocycle;

R⁹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

R²⁴ is -aryl, -heterocycle, or -carbocycle;

R²⁵ is —H, C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -carbocycle,—O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀ alkynyl), or OR¹⁸wherein R¹⁸ is —H, a hydroxyl protecting group, or a direct bond whereOR¹⁸ represents ═O;

R²⁶ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or -C₂-C₂₀ alkynyl, -aryl,-heterocycle, or -carbocycle;

R¹⁰ is -aryl or -heterocycle;

Z is —O, —S, —NH, or —NR¹², wherein R¹² is —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, or —C₂-C₂₀ alkynyl;

R¹¹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -aryl,-heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is —C₂-C₂₀ alkylene, -C₂-C₂₀ alkenylene, or —C₂-C₂₀ alkynylene;

R¹⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl;

each occurrence of R¹⁵ is independently —H, —COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl;

each occurrence of R¹⁶ is independently —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH; and

n is an integer ranging from 0 to 6;

wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,aryl, carbocyle, and heterocycle radicals, whether alone or as part ofanother group, are optionally substituted.

Auristatins of the formula D_(E) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted.

Auristatins of the formula D_(E) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R¹⁹,R²⁰ and R²¹ are optionally substituted as described herein.

Auristatins of the formula D_(E) include those wherein

R² is C₁-C₈ alkyl;

R³, R⁴ and R⁷ are independently selected from —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, carbocycle, aryl and heterocycle radicals areoptionally substituted;

R⁵ is —H;

R⁶ is —C₁-C₈ alkyl;

each R⁸ is independently selected from —OH, —O—(C₁-C₂₀ alkyl),—O—(C₂-C₂₀ alkenyl), or —O—(C₂-C₂₀ alkynyl) wherein said alkyl, alkenyl,and alkynyl radicals are optionally substituted;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is optionally substituted -phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is selected from —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀alkynyl, or -carbocycle; wherein said alkyl, alkenyl, alkynyl andcarbocycle radicals are optionally substituted; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein

R² is methyl;

R³ is —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, or C₂-C₈ alkynyl, wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted;

R⁴ is —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, monocyclic C₃-C₆carbocycle, —C₆-C₁₀ aryl, —C₁-C₈ alkylene(C₆-C₁₀ aryl), —C₂-C₈alkenylene(C₆-C₁₀ aryl), —C₂-C₈ alkynylene(C₆-C₁₀ aryl), —C₁-C₈ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₈ alkenylene (monocyclic C₃-C₆carbocycle), —C₂-C₈ alkynylene(monocyclic C₃-C₆ carbocycle); whereinsaid alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl andcarbocycle radicals whether alone or as part of another group areoptionally substituted;

R⁵ is —H;

R⁶ is methyl;

R⁷ is —C₁-C₈ alkyl, —C₂-C₈ alkenyl or —C₂-C₈ alkynyl;

each R⁸ is methoxy;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is -phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is methyl;

or a pharmaceutically acceptable salt form thereof.

Auristatins of the formula D_(E) include those wherein:

R² is methyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₅ alkyl; R⁵ is —H; R⁶is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H or—C₁-C₈ alkyl; R²⁴ is phenyl; R²⁵ is —OR¹⁸; wherein R¹⁸ is —H, a hydroxylprotecting group, or a direct bond where OR¹⁸ represents ═O; and R²⁶ ismethyl; or a pharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein:

R² is methyl or C₁-C₃ alkyl,

R³ is —H or —C₁-C₃ alkyl;

R⁴ is —C₁-C₅ alkyl;

R⁵ is H;

R⁶ is C1-C3 alkyl;

R⁷ is —C₁-C₅ alkyl;

R⁸ is —C₁-C₃ alkoxy;

R⁹ is —H or —C₁-C₈ alkyl;

R²⁴ is phenyl;

R²⁵ is —OR¹⁸; wherein R¹⁸ is —H, a hydroxyl protecting group, or adirect bond where OR¹⁸ represents ═O; and

R²⁶ is —C₁-C₃ alkyl;

or a pharmaceutically acceptable salt form thereof.

Auristatins of the formula D_(F) include those wherein

R² is methyl;

R³, R⁴, and R⁷ are independently selected from —H, —C₁-C₂₀ alkyl,—C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),—C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkynylene, carbocycle, aryl and heterocycle radicalswhether alone or as part of another group are optionally substituted;

R⁵ is —H;

R⁶ is methyl;

each R⁸ is methoxy;

R⁹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl; whereinsaid alkyl, alkenyl and alkynyl radical are optionally substituted;

R¹⁰ is optionally substituted aryl or optionally substitutedheterocycle;

Z is —O—, —S—, —NH—, or —NR¹², wherein R¹² is —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, or —C₂-C₂₀ alkynyl, each of which is optionally substituted;

R¹¹ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, -aryl,-heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂, wherein saidalkyl, alkenyl, alkynyl, aryl and heterocycle radicals are optionallysubstituted;

m is an integer ranging from 1-1000 or m=0;

R¹³ is —C₂-C₂₀ alkylene, —C₂-C₂₀ alkenylene, or —C₂-C₂₀ alkynylene, eachof which is optionally substituted;

R¹⁴ is —H, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, or —C₂-C₂₀ alkynyl whereinsaid alkyl, alkenyl and alkynyl radicals are optionally substituted;

each occurrence of R¹⁵ is independently —H, —COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl wherein said alkyl, alkenyland alkynyl radicals are optionally substituted;

each occurrence of R¹⁶ is independently —H, —C₁-C₂₀ alkyl, —C₂-C₂₀alkenyl, —C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH wherein said alkyl, alkenyland alkynyl radicals are optionally substituted;

n is an integer ranging from 0 to 6;

or a pharmaceutically acceptable salt thereof.

In certain of these embodiments, R¹⁰ is optionally substituted phenyl.

Auristatins of the formula D_(F) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R¹⁰and R¹¹ are as described herein.

Auristatins of the formula D_(F) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted.

Auristatins of the formula D_(F) include those wherein

R² is —C₁-C₃ alkyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₅ alkyl; R⁵ is—H; R⁶ is —C₁-C₃ alkyl; R⁷ is —C₁-C₅ alkyl; R⁸ is —C₁-C₃ alkoxy; R⁹ is—H or —C₁-C₈ alkyl; R¹⁰ is optionally substituted phenyl; Z is —O—, —S—,or —NH—; R¹¹ is as defined herein; or a pharmaceutically acceptable saltthereof.

Auristatins of the formula D_(F) include those wherein

R² is methyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₅ alkyl; R⁵ is —H; R⁶is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H or—C₁-C₈ alkyl; R¹⁰ is optionally substituted phenyl; Z is —O—, —S—, or—NH—; and R¹¹ is as defined herein; or a pharmaceutically acceptablesalt thereof.

Auristatins of the formula D_(F) include those wherein

R² is methyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₅ alkyl; R⁵ is —H; R⁶is methyl; R⁷ isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is —H or C₁-C₈alkyl; R¹⁰ is phenyl; and Z is —O— or —NH— and R¹¹ is as defined herein,preferably hydrogen; or a pharmaceutically acceptable salt form thereof.

Auristatins of the formula D_(F) include those wherein

R² is —C₁-C₃ alkyl; R³ is —H or —C₁-C₃ alkyl; R⁴ is —C₁-C₅ alkyl; R⁵ is—H; R⁶ is —C₁-C₃ alkyl; R⁷ is —C₁-C₅ alkyl; R⁸ is —C₁-C₃ alkoxy; R⁹ is—H or —C₁-C₈ alkyl; R¹⁰ is phenyl; and Z is —O— or —NH— and R¹¹ is asdefined herein, preferably hydrogen; or a pharmaceutically acceptablesalt form thereof.

Auristatins of the formula D_(E) or D_(F) include those wherein R³, R⁴and R⁷ are independently isopropyl or sec-butyl and R⁵ is —H. In anexemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is H, and R⁷ issec-butyl. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(E) or D_(F) include those wherein R² andR⁶ are each methyl, and R⁹ is H. The remainder of the substituents areas defined herein.

Auristatins of the formula D_(E) or D_(F) include those wherein eachoccurrence of R⁸ is —OCH₃. The remainder of the substituents are asdefined herein.

Auristatins of the formula D_(E) or D_(F) include those wherein R³ andR⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is H, R⁷ issec-butyl, each occurrence of R⁸ is —OCH₃, and R⁹ is H. The remainder ofthe substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein Z is —O— or —NH—.The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein R¹⁰ is aryl. Theremainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein R¹⁰ is -phenyl.The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) include those wherein Z is —O—, and R¹¹is H, methyl or t-butyl. The remainder of the substituents are asdefined herein.

Auristatins of the formula D_(F) include those wherein, when Z is —NH—,R¹¹ is —(O)_(m)—CH(R¹⁵)₂, wherein R¹⁵ is —(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is—C₁-C₈ alkyl or —(CH₂)_(n)—COOH. The remainder of the substituents areas defined herein.

Auristatins of the formula D_(F) include those wherein when Z is —NH—,R¹¹ is —(R¹³O)_(m)—CH(R¹⁵)₂, wherein R¹⁵ is —(CH₂)_(n)—SO₃H. Theremainder of the substitutents are as defined herein.

In preferred embodiments, when D is an auristatin of formula D_(E), w isan integer ranging from 1 to 12, preferably 2 to 12, y is 1 or 2, and ais preferably 1.

In some embodiments, wherein D is an auristatin of formula D_(F), a is 1and w and y are 0.

Illustrative Drug units (-D) include the drug units having the followingstructures:

or pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited totriethylene glycol esters (TEG) can be attached to the Drug Unit at R¹¹.Without being bound by theory, the hydrophilic groups assist in theinternalization and non-agglomeration of the Drug Unit.

In some embodiments, the Drug unit is not TZT-1027. In some embodiments,the Drug unit is not auristatin E, dolastatin 10, or auristatin PE.

In preferred embodiment, antibody-drug conjugate compounds have thefollowing structures wherein “L” or “mAb-s-” represents a 161P2F10B MAbdesignated H16-7.8 set forth herein:

-   L-MC-vc-PAB-MMAF-   or

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-   L-MC-vc-PAB-MMAE-   or

-   L-MC-MMAF

or pharmaceutically acceptable salt thereof.

In some embodiments, the Drug Unit is a calicheamicin, camptothecin, amaytansinoid, or an anthracycline. In some embodiments the drug is ataxane, a topoisomerase inhibitor, a vinca alkaloid, or the like.

In some typical embodiments, suitable cytotoxic agents include, forexample, DNA minor groove binders (e.g., enediynes and lexitropsins, aCBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, and vinca alkaloids. Othercytotoxic agents include, for example, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In some embodiments, the Drug is an anti-tubulin agent. Examples ofanti-tubulin agents include, auristatins, taxanes (e.g., Taxol®(paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids(e.g., vincristine, vinblastine, vindesine, and vinorelbine). Otherantitubulin agents include, for example, baccatin derivatives, taxaneanalogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid,estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins,discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131).

In certain embodiments, the cytotoxic or cytostatic agent is adolastatin. In certain embodiments, the cytotoxic or cytostatic agent isof the auristatin class. Thus, in a specific embodiment, the cytotoxicor cytostatic agent is MMAE (Formula XI). In another specificembodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI).

In certain embodiments, the cytotoxic or cytostatic agent is a compoundof formulas XII-XXI or pharmaceutically acceptable salt thereof:

p is 1 to 12. In more preferred embodiment, antibody-drug conjugatecompounds have the following structures wherein “mAb-s-” represents an161P2F10B MAb designated H16-7.8 set forth herein, and p is 4:

In a specific embodiment, the cytotoxic or cytostatic agent is MMAF(Formula XVIV).

X.) Drug Loading

Drug loading is represented by p and is the average number of drugmoieties per antibody in a molecule. Drug loading may range from 1 to 20drug moieties (D) per antibody. ADCs of the invention includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy and, ELISA assay. Thequantitative distribution of ADC in terms of p may also be determined Insome instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g., p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 12; from 1 to about 8; from about 2 to about 6; from about 3 toabout 5; from about 3 to about 4; from about 3.1 to about 3.9; fromabout 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 toabout 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.Indeed, it has been shown that for certain ADCs, the optimal ratio ofdrug moieties per antibody may be less than 8, and may be about 2 toabout 5. See US 2005-0238649 A1 (herein incorporated by reference in itsentirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g., hydrophobic interaction chromatography (see, e.g., Hamblett, K.J., et al. “Effect of drug loading on the pharmacology,pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,”Abstract No. 624, American Association for Cancer Research, 2004 AnnualMeeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March2004; Alley, S. C., et al. “Controlling the location of drug attachmentin antibody-drug conjugates,” Abstract No. 627, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004). In certain embodiments, a homogeneousADC with a single loading value may be isolated from the conjugationmixture by electrophoresis or chromatography.

XI.) Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of a Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of ³H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that a Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Page,et al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytotoxicity (Skehan, et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, No. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza, et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a 161P2F10B therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic cancermodels can be used, wherein cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice (Klein, et al., 1997, Nature Medicine 3: 402-408). Forexample, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540describe various xenograft models of human prostate cancer capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease. Efficacy can be predicted using assays that measure inhibitionof tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XII.) Treatment of Cancer(s)

The identification of 161P2F10B as a protein that is normally expressedin a restricted set of tissues, but which is also expressed in cancerssuch as those listed in Table I, opens a number of therapeuticapproaches to the treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when thetargeted protein is expressed on normal tissues, even vital normal organtissues. A vital organ is one that is necessary to sustain life, such asthe heart or colon. A non-vital organ is one that can be removedwhereupon the individual is still able to survive. Examples of non-vitalorgans are ovary, breast, and prostate.

Expression of a target protein in normal tissue, even vital normaltissue, does not defeat the utility of a targeting agent for the proteinas a therapeutic for certain tumors in which the protein is alsooverexpressed. For example, expression in vital organs is not in and ofitself detrimental. In addition, organs regarded as dispensable, such asthe prostate and ovary, can be removed without affecting mortality.Finally, some vital organs are not affected by normal organ expressionbecause of an immunoprivilege Immunoprivileged organs are organs thatare protected from blood by a blood-organ barrier and thus are notaccessible to immunotherapy. Examples of immunoprivileged organs are thebrain and testis.

Accordingly, therapeutic approaches that inhibit the activity of a161P2F10B protein are useful for patients suffering from a cancer thatexpresses 161P2F10B. These therapeutic approaches generally fall intothree classes. The first class modulates 161P2F10B function as itrelates to tumor cell growth leading to inhibition or retardation oftumor cell growth or inducing its killing. The second class comprisesvarious methods for inhibiting the binding or association of a 161P2F10Bprotein with its binding partner or with other proteins. The third classcomprises a variety of methods for inhibiting the transcription of a161P2F10B gene or translation of 161P2F10B mRNA.

Accordingly, Cancer patients can be evaluated for the presence and levelof 161P2F10B expression, preferably using immunohistochemicalassessments of tumor tissue, quantitative 161P2F10B imaging, or othertechniques that reliably indicate the presence and degree of 161P2F10Bexpression Immunohistochemical analysis of tumor biopsies or surgicalspecimens is preferred for this purpose. Methods for immunohistochemicalanalysis of tumor tissues are well known in the art.

XIII) 161P2F10B as a Target for Antibody-Based Therapy

161P2F10B is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 161P2F10B is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of161P2F10B-immunoreactive compositions are prepared that exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunoreactive composition tonon-target organs and tissues. Antibodies specifically reactive withdomains of 161P2F10B are useful to treat cancers (preferably cancer ofTable I, more preferably kidney and/or liver cancer) systemically,preferably as antibody drug conjugates (i.e., ADCs) wherein theconjugate is with a toxin or therapeutic agent.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 161P2F10B sequence shown in FIG. 1. In addition,skilled artisans understand that it is routine to conjugate antibodiesto cytotoxic agents (see, e.g., Slevers, et al., Blood 93:11 3678-3684(Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivereddirectly to cells, such as by conjugating them to antibodies specificfor a molecule expressed by that cell (e.g., 161P2F10B), the cytotoxicagent will exert its known biological effect (i.e., cytotoxicity) onthose cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an mammal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g., a 161P2F10B MAb, preferably H16-7.8) that binds to an antigen(e.g., 161P2F10B) expressed, accessible to binding or localized on thecell surfaces. A typical embodiment is a method of delivering acytotoxic and/or therapeutic agent to a cell expressing 161P2F10B,comprising conjugating the cytotoxic agent to an antibody thatimmunospecifically binds to a 161P2F10B epitope, and, exposing the cellto the antibody drug conjugate (ADC). Another illustrative embodiment isa method of treating an individual suspected of suffering frommetastasized cancer, comprising a step of administering parenterally tosaid individual a pharmaceutical composition comprising atherapeutically effective amount of an antibody conjugated to acytotoxic and/or therapeutic agent.

Cancer immunotherapy using 161P2F10B antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen, et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki, et al., 1997, Blood 90:3179-3186, Tsunenari, etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk, et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi, et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong, et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun, et al., 1994,Cancer Res. 54:6160-6166; Velders, et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard, et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™ Coulter Pharmaceuticals) respectively,while others involve co-administration of antibodies and othertherapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel(Genentech, Inc.).

In a preferred embodiment, the antibodies will be conjugated a cytotoxicagent, supra, preferably an auristatin derivative designated MMAF(Seattle Genetics).

Preferred monoclonal antibodies used in the therapeutic methods of theinvention are those that are either fully human and that bindspecifically to the target 161P2F10B antigen with high affinity.

XIV.) 161P2F10B ADC Cocktails

Therapeutic methods of the invention contemplate the administration ofsingle 161P2F10B ADCs as well as combinations, or cocktails, ofdifferent MAbs (i.e., 161P2F10B MAbs or Mabs that bind another protein).Such MAb cocktails can have certain advantages inasmuch as they containMAbs that target different epitopes, exploit different effectormechanisms or combine directly cytotoxic MAbs with MAbs that rely onimmune effector functionality. Such MAbs in combination can exhibitsynergistic therapeutic effects. In addition, 161P2F10B MAbs can beadministered concomitantly with other therapeutic modalities, includingbut not limited to various chemotherapeutic and biologic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. In a preferred embodiment, the 161P2F10B MAbs areadministered in conjugated form.

161P2F10B ADC formulations are administered via any route capable ofdelivering the antibodies to a tumor cell. Routes of administrationinclude, but are not limited to, intravenous, intraperitoneal,intramuscular, intratumor, intradermal, and the like. Treatmentgenerally involves repeated administration of the 161P2F10B ADCpreparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range, includingbut not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general,doses in the range of 10-1000 mg MAb per week are effective and welltolerated.

Based on clinical experience with the Herceptin® (trastuzumab) in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the MAb preparation represents an acceptabledosing regimen. Preferably, the initial loading dose is administered asa 90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the MAbs used, the degree of 161P2F10B expression in thepatient, the extent of circulating shed 161P2F10B antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of 161P2F10B ina given sample (e.g., the levels of circulating 161P2F10B antigen and/or161P2F10B expressing cells) in order to assist in the determination ofthe most effective dosing regimen, etc. Such evaluations are also usedfor monitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

An object of the present invention is to provide 161P2F10B ADCs, whichinhibit or retard the growth of tumor cells, preferably tumor cells ofTable I. A further object of this invention is to provide methods toinhibit angiogenesis and other biological functions and thereby reducetumor growth in mammals, preferably humans, using such 161P2F10B ADCs,and in particular using such 161P2F10B ADCs combined with other drugs orimmunologically active treatments.

XV.) Combination Therapy

In one embodiment, there is synergy when tumors, including human tumors,are treated with 161P2F10B ADCs in conjunction with chemotherapeuticagents or radiation or combinations thereof. In other words, theinhibition of tumor growth by a 161P2F10B ADC is enhanced more thanexpected when combined with chemotherapeutic agents or radiation orcombinations thereof. Synergy may be shown, for example, by greaterinhibition of tumor growth with combined treatment than would beexpected from a treatment of only 161P2F10B ADC or the additive effectof treatment with a 161P2F10B ADC and a chemotherapeutic agent orradiation. Preferably, synergy is demonstrated by remission of thecancer where remission is not expected from treatment either from a161P2F10B ADC or with treatment using an additive combination of a161P2F10B ADC and a chemotherapeutic agent or radiation.

The method for inhibiting growth of tumor cells using a 161P2F10B ADCand a combination of chemotherapy or radiation or both comprisesadministering the 161P2F10B ADC before, during, or after commencingchemotherapy or radiation therapy, as well as any combination thereof(i.e., before and during, before and after, during and after, or before,during, and after commencing the chemotherapy and/or radiation therapy).For example, the 161P2F10B ADC is typically administered between 1 and60 days, preferably between 3 and 40 days, more preferably between 5 and12 days before commencing radiation therapy and/or chemotherapy.However, depending on the treatment protocol and the specific patientneeds, the method is performed in a manner that will provide the mostefficacious treatment and ultimately prolong the life of the patient.

The administration of chemotherapeutic agents can be accomplished in avariety of ways including systemically by the parenteral and enteralroutes. In one embodiment, the 161P2F10B ADCs and the chemotherapeuticagent are administered as separate molecules. Particular examples ofchemotherapeutic agents or chemotherapy include cisplatin, dacarbazine(DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin(adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine,etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine,bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin,asparaginase, busulfan, carboplatin, cladribine, dacarbazine,floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha,leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin,tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracilmustard, vinorelbine, chlorambucil, taxol and combinations thereof.

The source of radiation, used in combination with a 161P2F10B ADC, canbe either external or internal to the patient being treated. When thesource is external to the patient, the therapy is known as external beamradiation therapy (EBRT). When the source of radiation is internal tothe patient, the treatment is called brachytherapy (BT).

The above described therapeutic regimens may be further combined withadditional cancer treating agents and/or regimes, for example additionalchemotherapy, cancer vaccines, signal transduction inhibitors, agentsuseful in treating abnormal cell growth or cancer, antibodies (e.g.,Anti-CTLA-4 antibodies as described in WO2005/092380 (Pfizer)) or otherligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

When the mammal is subjected to additional chemotherapy,chemotherapeutic agents described above may be used. Additionally,growth factor inhibitors, biological response modifiers, anti-hormonaltherapy, selective estrogen receptor modulators (SERMs), angiogenesisinhibitors, and anti-androgens may be used. For example, anti-hormones,for example anti-estrogens such as Nolvadex (tamoxifen) or,anti-androgens such as Casodex(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-′-(trifluoromethyl)propionanilidemay be used.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

XVI.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise an antibody that is or can bedetectably labeled. Kits can comprise a container comprising a DrugUnit. The kit can include all or part of the amino acid sequences inFIG. 2, or FIG. 3 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a cancer of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as antibody drug conjugates (ADCs), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of cancers of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass, metal or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), cell population(s) and/or antibody(s). In anotherembodiment a container also comprises an antibody, binding fragmentthereof or specific binding protein for use in evaluating proteinexpression of 161P2F10B in cells and tissues, or for relevantlaboratory, prognostic, diagnostic, prophylactic and therapeuticpurposes; indications and/or directions for such uses can be included onor with such container, as can reagents and other compositions or toolsused for these purposes.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody drug conjugate specifically binding to 161P2F10B.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1 The 161P2F10B Antigen

The 161P2F10B gene sequence was discovered using Suppression SubtractiveHybridization (SSH) methods known in the art. The 161P2F10B SSH sequenceof 182 by was identified from cDNA derived from kidney cancer patientsusing standard methods. A full length cDNA clone for 161P2F10B wasisolated from kidney cancer patient specimens. The cDNA is 3858 by inlength and encodes a 875 amino acid ORF (See, FIG. 1). 161P2F10B showedhomology to ENPP3 (See, Buhring, et. al., Blood 97:3303-3305 (2001).161P2F10B maps to chromosome 6q22 using standard methods known in theart. For further reference see, U.S. Pat. No. 7,279,556 (Agensys, Inc.,Santa Monica, Calif.), U.S. Pat. No. 7,405,290 (Agensys, Inc., SantaMonica, Calif.), U.S. Pat. No. 7,067,130 (Agensys, Inc., Santa Monica,Calif.), and U.S. Pat. No. 7,226,594 (Agensys, Inc., Santa Monica,Calif.).

Example 2 Generation of 161P2F10B Monoclonal Antibodies (MAbs)

In one embodiment, therapeutic Monoclonal Antibodies (“MAbs”) to161P2F10B comprise those that react with epitopes specific for proteinthat would bind, internalize, disrupt or modulate the biologicalfunction of 161P2F10B, for example, those that would disrupt theinteraction with ligands, substrates, and binding partners Immunogensfor generation of such MAbs include those designed to encode or containthe extracellular domains or the entire 161P2F10B protein sequence, andregions predicted to contain functional motifs predicted to be antigenicfrom computer analysis of the amino acid sequence Immunogens includepeptides and recombinant proteins such as tag5-161P2F10B, a purifiedmammalian cell derived His tagged protein. In addition, cells engineeredthrough retroviral transduction to express high levels of 161P2F10B,such as RAT1-161P2F10B, are used to immunize mice.

MAbs to 161P2F10B were generated using XenoMouse® technology (AmgenFremont) wherein the murine heavy and kappa light chain loci have beeninactivated and a majority of the human heavy and kappa light chainimmunoglobulin loci have been inserted. The MAb designated H16-7.8 wasgenerated from immunization with human γ2 producing XenoMice withTagS-161P2F10B cells.

The 161P2F10B MAb H16-7.8 specifically binds to recombinant 161P2F10B(SEQ ID NO:2) expressing cells and endogenous cell surface 161P2F10Bexpressed in cancer xenograft cells.

DNA coding sequences for 161P2F10B MAb H16-7.8 were determined afterisolating mRNA from the respective hybridoma cells with TRIzol® reagent(Life Technologies, Gibco BRL).

Anti-161P2F10B H16-7.8 heavy and light chain variable nucleic acidsequences were sequenced from the hybridoma cells using the followingprotocol. H16-7.8 secreting hybridoma cells were lysed with TRIzol®reagent (Life Technologies, Gibco BRL). Total RNA was purified andquantified. First strand cDNAs was generated from total RNA with oligo(dT)12-18 priming using the Gibco®-BRL Superscript Preamplificationsystem. First strand cDNA was amplified using human immunoglobulinvariable heavy chain primers, and human immunoglobulin variable lightchain primers. PCR products were sequenced and the variable heavy andlight chain regions determined

The nucleic acid and amino acid sequences of the variable heavy andlight chain regions are listed in FIG. 2 and FIG. 3. The heavy chainvariable region of H16-7.8 consists of the amino acid sequence rangingfrom 20^(th) Q residue to the 142^(nd) S residue of SEQ ID NO:7, and thelight chain variable region of H16-7.8 consists of the amino acidsequence ranging from 20^(th) E residue to the 127^(th) R residue of SEQID NO:8. The heavy chain of H16-7.8 consists of the amino acid sequenceranging from 20^(th) Q residue to the 468^(th) K residue of SEQ ID NO:7and the light chain of H16-7.8 consists of amino acid sequence rangingfrom 20^(th) E residue to the 233^(th) C residue of SEQ ID NO:8.Alignment of H16-7.8 to human VH4-31/D5-12/JH6 germline and humanA26/JK1 germline is set forth in FIG. 4A-4B.

Example 3 Expression of H16-7.8 Using Recombinant DNA Methods

To express H16-7.8 recombinantly in transfected cells, H16-7.8 heavy andlight chain sequences (SEQ ID NO:7, from 20 to 468 and the light chainof SEQ ID NO:8, from 20 to 233) were cloned into expression vectors. Thecomplete H16-7.8 human heavy chain and light chain cassettes were cloneddownstream of the CMV promoter/enhancer in a cloning vector. Apolyadenylation site was included downstream of the MAb coding sequence.The recombinant H16-7.8 expressing constructs were transfected into CHOcells and recombinant H16-7.8 were secreted from CHO cells. IgG titerswere measured by ELISA. Results confirmed IgG and expression and goodco-expression of the heavy and light chains. Recombinant H16-7.8 wereevaluated for binding to cell surface 161P2F10B by flow cytometry (FIG.5). 3T3-control and 3T3-161P2F10B cells were stained with recombinantH16-7.8 from either hybridoma or from CHO cells transfected with H16-7.8heavy and light chain vector constructs.

Binding was detected by flow cytometry. Results show that therecombinantly expressed H16-7.8 in CHO cells is secreted and bindsspecifically to cell surface 161P2F10B. (FIG. 5).

Recombinant H16-7.8 was characterized with respect to its peptidesequence. The peptide mapping analysis of H16-7.8 confirmed that thededuced amino acid sequence of H16-7.8 is correct versus the sequencedetermined using Lys-C digestion with LC-MS/MS, Asp-N digestion withLC-MS/MS, and N-terminal sequence by Edman degradation.

Example 4 Antibody Drug Conjugation of H16-7.8

The H16-7.8 (FIG. 2) was conjugated to adolavaline-valine-dolaisoleucine-dolaproine-phenylalanine (aurastatinderivative) designated MMAF (Formula XVIV; monomethyl auristatin F)using a maleimidocaproyl (mc) non-cleavble linker set forth below:

The synthesis of the maleimidocaproyl (mc) non-cleavable linker to theMMAF (Seattle Genetics, Seattle, Wash.) was completed (SAFC, Madison,Wis.) using the synthesis method set forth in Table VI to create thecytotoxic mcMMAF.

Then, the antibody drug conjugate (ADC) of the invention designatedH16-7.8mcMMAF was made using the following protocols.

Briefly, a 10 mg/mL solution of the H16-7.8 in 10 mM succinate, at pH4.5 was buffer exchanged by diafiltration. The purpose of the bufferexchange is to remove H16-7.8 formulation buffer components and replaceit with a more compatible buffer that is optimized for the subsequent“reduction” step. The antibody was diafiltered against 6 diavolumes (DV)of sodium borate buffer and concentrated to 10±1 mg/ml, flushed from thesystem, diluted to 7.5 mg/ml and 0.2 μm filtered.

Subsequently, EDTA was added to 5 mM final concentration in the reactionmixture. Next, the disulfide bonds of the H16-7.8 were partially reducedwith tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) to form freethiols (SH). This process is performed at 37° C. EDTA was present at 5mM concentration during this reaction to chelate any divalent metalcations that could cause unwanted SH re-oxidation. At the end of thereduction step the temperature of the reaction solution was lowered to20° C. and analyzed to determine the molar ratio of free SH and toensure that ≧3.9 SH per MAb had been generated. For conjugation, thedrug-linker mcMMAF was weighed out in the isolator and dissolved in DMSOto a concentration of 5.5 mg/ml.

The SH groups on the partially reduced H16-7.8 were reacted withdrug-linker mcMMAF to form the conjugate, H16-7.8mcMMAF. The mcMMAF inDMSO was added at a set molar equivalent of drug to antibody. This stepwas performed at 20° C. After 1 h incubation period any excess of thedrug-linker in the reaction was quenched with N-Acetyl- cystein, thuseliminating any reactive drug-linker and turning it into and adduct thatis easier to remove and to detect by analytical methods. The mixture isthen stirred for fifteen (15) additional minutes following the additionof one (1) molar equivalents of N-Acetyl-Cysteine relative to mcMMAF.

Next, ultrafiltration/dialfiltration is performed in order to removeDMSO, process impurities, and buffer exchange the ADC into formulationbuffer.

Excess quenched mcMMAF are thus removed by ultrafiltration/diafiltrationof the antibody drug conjugate (ADC) with 8 diavolumes of 20 mMhistidine, pH 5.2 formulation buffer. After the completion of the 8thdiavolume, the solution is recirculated and assayed for proteinconcentration.

The conjugate is adjusted to six (6)±0.5 mg/ml with 20 mM Histidine, 10%trehalose, pH 5.2 buffer. Polysorbate 20 is then added, mixed tohomogeneity, and aseptically filtered through a 0.2 μm filter.

The resulting antibody drug conjugate (ADC) is designated H16-7.8mcMMAFand has the following formula:

wherein MAb is H16-7.8 (FIG. 2 and FIG. 3) and p is from 1 to 12.

Example 5 Characterization of H16-7.8 and H16-7.8mcMMAF

The H16-7.8 was generated using the procedures set forth in the exampleentitled Example 2 “Generation of 161P2F10B Monoclonal Antibodies(MAbs)”. Additionally, the Antibody Drug Conjugates that bind 161P2F10Bwere generated using the procedures set forth in the example entitled“Antibody Drug Conjugation of H16-7.8”. The H16-7.8 and H16-7.8mcMMAFADC were screened, identified, and characterized using a combination ofassays known in the art.

A. Cell Binding and Affinity Determination by FACS

H16-7.8 and H16-7.8mcMMAF were tested for the binding affinity to161P2F10B endogenously expressed on Ku812 cells. Briefly, eleven (11)dilutions of H16-7.8 or H16-7.8mcMMAF are incubated with Ku812 cells(50,000 cells per well) overnight at 4° C. at a final concentration of160 nM to 0.0001 nM. At the end of the incubation, cells are washed andincubated with anti-hIgG-PE detection antibody for 45 min at 4° C. Afterwashing the unbound detection antibodies, the cells are analyzed byFACS. Mean Florescence Intensity (MFI) values are obtained (See, TableIV). MFI values were entered into Graphpad Prisim software and analyzedusing the one site binding (hyperbola) equation of Y=Bmax*X/(Kd+X) togenerate H16-7.8 or H16-7.8mcMMAF saturation curves shown in FIG. 6.Bmax is the MFI value at maximal binding of H16-7.8 or H16-7.8mcMMAF to161P2F10B; Kd is H16-7.8 or H16-7.8mcMMAF binding affinity which is theconcentration of H16-7.8 or H16-7.8mcMMAF required to reach half-maximalbinding.

The calculated affinity (Kd) of H16-7.8 and H16-7.8mcMMAF is 0.06 nM and0.19 nM, respectively on 161P2F10B-related protein endogenouslyexpressed on the surface of Ku812 cells.

To determine binding of H16-7.8 and H16-7.8mcMMAF to endogenous161P2F10B-related protein expressed on the surface of renal cancer cellshuman UGK-3 cells (patient derived clear cell renal cancer) and RXF-393cells (clear cell renal cancer) were stained with 10 μg/ml of nativeH16-7.8, H16-7.8mcMMAF, or an isotype control human IgG2 and evaluatedby FACS.

The results in FIG. 7 (left panels) demonstrate strong staining of thetwo different renal tumor cells with H16-7.8 (gray lines), but not withthe control MAb (filled histograms). The panels on the right demonstratea similar strong staining of the same renal tumor cells withH16-7.8mcMMAF (gray lines). (FIG. 7; right panels). These results showthat both H16-7.8 and H16-7.8mcMMAF bind native 161P2F10B antigenexpressed on the surface of human cancer cells. Conjugation of nativeH16-7.8 to generate the H16-7.8mcMMAF did not alter its cell surfacebinding to native 161P2F10B antigen expressed on human cancer cells.

Example 6 Cell Cytotoxicity Mediated by H16-7.8mcMMAF

The ability of H16-7.8mcMMAF to mediate 161P2F10B-dependent cytotoxicitywas evaluated in KU812 cells engineered to express 161P2F10B. For thisassay 2000 viable KU812 cells were plated in triplicate on Day 0 andallowed to recover overnight. The next day, serial 1:4 dilutions ofdifferent lots of H16-7.8mcMMAF or a control MAb conjugated with mcMMAFwas added to yield the final concentrations indicated in FIG. 8. Thecells were allowed to incubate for six (6) days at which time 20 μl ofAlamar blue was added to each well. The plates were incubated for anadditional four (4) hours and the fluorescence intensity read on afluorescent plate reader using an excitation wavelength of 540 nM and anemission wavelength of 620 nM.

The results in FIG. 8 show that both lots of H16-7.8mcMMAF potentlyinhibited the proliferation of KU812 cells. The IC 50 was determined tobe 0.2 nM and 0.1 nM for Lot (1) and Lot (2) respectively. A fully humanControl MAb that does not bind KU812 cells was conjugated with mcMMAF toyield a DAR of 3.9 (+/−0.2). The Control ADC did not inhibit KU812 cellproliferation further demonstrating the specificity of cytotoxicity.Thus, these results indicate that H16-7.8mcMMAF can selectively delivera cytotoxic drug to 161P2F10B expressing cells leading to their killing.

Example 7 H16-7.8mcMMAF Inhibit Growth of Tumors In Vivo

The significant expression of 161P2F10B on the cell surface of tumortissues, together with its restrictive expression in normal tissuesmakes 161P2F10B a good target for antibody therapy and similarly,therapy via ADC. Thus, the therapeutic efficacy of H16-7.8mcMMAF inhuman kidney cancer xenograft mouse models is evaluated.

Antibody drug conjugate efficacy on tumor growth and metastasisformation is studied in mouse cancer xenograft models (e.g.,subcutaneous and orthotopically).

Subcutaneous (s.c.) tumors are generated by injection of 5×10⁴-10⁶cancer cells mixed at a 1:1 dilution with Matrigel (CollaborativeResearch) in the right flank of male SCID mice. To test ADC efficacy ontumor formation, i.e., ADC injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified human IgG or PBS; or a purified MAb that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between control IgG or PBS on tumor growth. Tumorsizes are determined by caliper measurements, and the tumor volume iscalculated as length×width×height. Mice with subcutaneous tumors greaterthan 1.5 cm in diameter are sacrificed.

Growth of kidney tumors in mice are performed by injection of 1.5million to 2 million cells implanted subcutaneously into male SCID mice.Mice are monitored for general health, physical activity, and appearanceuntil they become moribund. At the time of sacrifice, the mice can beexamined to determine tumor burden and other organs harvested toevaluate metastasis to distant sites. Alternatively, death can be usedas an endpoint. The mice are then segregated into groups for theappropriate treatments, with 161P2F10B or control MAbs beingadministered via i.v. injection.

An advantage of xenograft cancer models is the ability to studyneovascularization and angiogenesis. Tumor growth is partly dependent onnew blood vessel development. Although the capillary system anddeveloping blood network is of host origin, the initiation andarchitecture of the neovasculature is regulated by the xenograft tumor(Davidoff, et al., Clin Cancer Res. (2001) 7:2870; Solesvik, et al., EurJ Cancer Clin Oncol. (1984) 20:1295). The effect of antibody and smallmolecule on neovascularization is studied in accordance with proceduresknown in the art, such as by IHC analysis of tumor tissues and theirsurrounding microenvironment.

H16-7.8mcMMAF inhibits formation of kidney cancer xenografts. Theseresults indicate the utility of H16-7.8mcMMAF in the treatment of localand advanced stages of cancer and preferably those cancers set forth inTable I.

161P2F10B ADCs:

Monoclonal antibodies were raised against 161P2F10B as described in theExample entitled “Generation of 161P2F10B Monoclonal Antibodies (MAbs).”Further the MAbs are conjugated to a toxin as described in the Exampleentitled “Antibody Drug Conjugation of H16-7.8” to form H16-7.8mcMMAF.The H16-7.8mcMMAF is characterized by FACS, and other methods known inthe art to determine its capacity to bind 161P2F10B.

Cell Lines and Xenografts:

The RFX-393 cells are maintained in RPMI, supplemented with L-glutamineand 10% FBS. UG-K3 and SKRC-01 xenografts are maintained by serialpropagation in SCID mice.

Efficacy of H16-7.8mcMMAF in Subcutaneously Established Human RenalCancer Xenograft UG-K3 in SCID Mice.

In this experiment, patient-derived human renal cancer xenograft UG-K3was maintained by serial passages in SCID mice. Stock tumors wereharvested sterilely and minced to small pieces. The tumor pieces wereenzymatically digested to single cell suspensions using LiberaseBlendzyme (Roche Applied Science, Indianapolis, Ind.). 1.5×10⁶ cellswere injected into the flanks of individual SCID mice and tumors wereallowed to grow untreated until they reached an approximate volume of100 mm³ Animals were randomly assigned to the following cohorts: anH16-7.8mcMMAF treated group, an H16-7.8 control and a 5% Dextrosecontrol. H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once on day 0by intravenous bolus injection. The amount of H16-7.8mcMMAF and H16-7.8administered was based on the individual body weight of each animalobtained immediately prior to dosing. The 5% Dextrose control was dosedat 150 μL per animal. Tumor growth was monitored using calipermeasurements every 3 to 4 days until the end of the study. Tumor volumeis calculated as Width×Length/2, where width is the smallest dimensionand length is the largest. Animals in control groups were humanelyeuthanized when tumors reached approximately 1000 mm³. Animals inH16-7.8mcMMAF treated group were monitored for an additional two weeksbefore sacrifice. Statistical analysis was performed at the last timepoint when data for both control groups were available, usingKruskal-Wallis test with a=0.05.

The results demonstrated that treatment of UG-K3 renal clear cellxenograft tumors with H16-7.8mcMMAF at all doses and schedules examinedresulted in significant inhibition of tumor growth in SCID mice. (FIG.9).

Growth Inhibition of Established Orthotopic UG-K3 Xenografts byH16-7.8mcMMAF

In this experiment, the ability of H16-7.8mcMMAF to inhibit the growthof established renal tumors grown orthotopically was evaluated usingpatient-derived, UG-K3 tumor xenografts. Briefly, stocks of UG-K3 tumorswere digested enzymatically and 1.5 million viable cells were surgicallyimplanted into the kidneys of male SCID mice on Day 0. The tumors wereallowed to grow for 7 days at which time animals were randomized to 4different treatment groups (n=10 per group). Animals randomized to GroupA received Control ADC at 5 mpk, Group B received H16-7.8mcMMAF at 3mg/kg and Group C received H16-7.8mcMMAF at 5 mg/kg administered every 4days for a total of 4 doses. Group D received H16-7.8mcMMAF at 10 mg/kgone time. At the end of the study (Day 41) the animals were sacrificedand the right and left kidneys weighed on an electronic balance. Thetumor weights plotted on the graph were determined by subtracting theweight of the tumor-free contralateral kidney from the weight of thetumor-bearing right kidney.

The results demonstrated that treatment of UG-K3 renal clear cellxenograft tumors with H16-7.8mcMMAF at all doses and schedules examinedresulted in dramatic inhibition of tumor growth (FIG. 10). Tumor weightsin all H16-7.8mcMMAF treatment groups (B, C, and D) were less than 1% ofthe tumor weights in the Control treated group. These differences werehighly statistically significant (p<0.0001, ANOVA).

Efficacy of H16-7.8mcMMAF in Subcutaneously Established Human RenalCancer Xenograft RXF-393 in SCID Mice.

In this experiment, human renal cancer cells RXF-393 (0.5×10⁶ cells permouse) were injected into the flanks of individual mice and tumors wereallowed to grow untreated until they reached an approximate volume of100 mm³ Animals were then randomly assigned to the following cohorts: anH16-7.8mcMMAF treated group, an H16-7.8 treated group and a 5% Dextrosecontrol. H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once a weekfor a total of two doses by intravenous bolus injection. The amount ofH16-7.8mcMMAF and H16-7.8 administered was based on the individual bodyweight of each animal obtained immediately prior to dosing. The 5%Dextrose control was dosed at 150 μL per animal. Tumor growth wasmonitored using caliper measurements every 3 to 4 days until the end ofthe study. Tumor volume is calculated as Width²×Length/2, where width isthe smallest dimension and length is the largest. Animals in controlgroups were humanely euthanized when tumors reached approximately 1000mm³. Animals in H16-7.8mcMMAF treated group were monitored for anadditional two weeks before sacrifice.

The results demonstrated that treatment of RFX-393 human renal cancerxenograft tumors with H16-7.8mcMMAF at all doses and schedules examined(including single dose) resulted in significant inhibition of tumorgrowth in SCID mice. Statistical analysis was performed at the last timepoint when data in both control groups were available, usingKruskal-Wallis test with α=0.05. (FIG. 11).

Efficacy Study of H16-7.8 Compared to H16-7.8mcMMAF in SubcutaneouslyEstablished Human Renal Cancer SKRC-01 in SCID Mice

In another experiment, human renal cancer cells SKRC-01 (0.8×10⁶ cellsper mouse) were injected into the flanks of individual mice. Tumors wereallowed to grow untreated until they reached an approximate volume of100 mm³. On day 0 when tumors reach 100 mm³, animals were randomlyassigned to the following cohorts: an H16-7.8mcMMAF treated group, anH16-7.8 treated group and a 5% Dextrose control. H16-7.8mcMMAF andH16-7.8 were dosed at 4 mg/kg every four days for a total of four dosesby intravenous bolus injection. The amount of H16-7.8mcMMAF and H16-7.8administered was based on the individual body weight of each animalobtained immediately prior to dosing. The 5% Dextrose control was dosedat 150 μL per animal. Tumor growth was monitored using calipermeasurements every 3 to 4 days. Tumor volume was calculated asWidth²×Length/2, where width is the smallest dimension and length is thelargest.

The results show that the ADC H16-7.8mcMMAF significantly inhibited thegrowth of SKRC-01 tumor formation at all doses (including single dose),while the naked MAb H16-7.8 had no effect. Thus, the ADC H16-7.8mcMMAFhad a significantly more prominent effect that the naked antibodyH16-7.8. (FIG. 12).

Efficacy Study of H16-7.8mcMMAF compared to other 161P2F10B AntibodyDrug Conjugates (ADCs) in Subcutaneous Established UG-K3 in SCID Mice

In another experiment, human renal cancer cells UG-K3 (1.5×10⁶ cells permouse) were injected into the flanks of individual mice. Tumors wereallowed to grow untreated until they reached an approximate volume of100 mm³ On day 0 when tumors reach 100 mm³, animals were randomlyassigned to the following cohorts: an H16-7.8mcMMAF, an H16-7.8vcMMAE,and H16-1.11mcMMAF, and H16-1.11vcMMAE, a PBS control, and a controlMAb-vcMMAE treated group. All antibody drug conjugates (ADCs) were dosedat 10 mg/kg once on day 0. The amount of each ADC administered was basedon the individual body weight of each animal obtained immediately priorto dosing. The PBS control was dosed at 150 μ/L per animal. Tumor growthwas monitored using caliper measurements every 3 to 4 days. Tumor volumewas calculated as Width×Length/2, where width is the smallest dimensionand length is the largest.

The results show that the ADCs H16-7.8vcMMAE and H16-1.11vcMMAE did notinhibit tumor formation growth. Additionally, both the H16-7.8mcMMAF andH16-1.11mcMMAF significantly inhibited the growth of UG-K3 tumorformation during the first thirty (30) days. After day thirty (30) theH16-7.8mcMMAF had a significantly more prominent effect when compared toH16-1.11mcMMAF. (FIG. 13).

Example 8 Human Clinical Trials for the Treatment and Diagnosis of HumanCarcinomas Through Use of 161P2F10B ADCs

161P2F10B ADCs are used in accordance with the present invention whichspecifically bind to 161P2F10B, and are used in the treatment of certaintumors, preferably those listed in Table I. In connection with each ofthese indications, two clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated with161P2F10B ADCs in combination with a chemotherapeutic or anti-neoplasticagent and/or radiation therapy or a combination thereof. Primary cancertargets, such as those listed in Table I, are treated under standardprotocols by the addition of 161P2F10B ADCs to standard first and secondline therapy. Protocol designs address effectiveness as assessed by thefollowing examples, including but not limited to, reduction in tumormass of primary or metastatic lesions, increased progression freesurvival, overall survival, improvement of patients health, diseasestabilization, as well as the ability to reduce usual doses of standardchemotherapy and other biologic agents. These dosage reductions allowadditional and/or prolonged therapy by reducing dose-related toxicity ofthe chemotherapeutic or biologic agent. 161P2F10B ADCs are utilized inseveral adjunctive clinical trials in combination with thechemotherapeutic or anti-neoplastic agents.

II.) Monotherapy: In connection with the use of the 161P2F10B ADCs inmonotherapy of tumors, the 161P2F10B ADCs are administered to patientswithout a chemotherapeutic or anti-neoplastic agent. In one embodiment,monotherapy is conducted clinically in end-stage cancer patients withextensive metastatic disease. Protocol designs address effectiveness asassessed by the following examples, including but not limited to,reduction in tumor mass of primary or metastatic lesions, increasedprogression free survival, overall survival, improvement of patientshealth, disease stabilization, as well as the ability to reduce usualdoses of standard chemotherapy and other biologic agents.

Dosage

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the antibody and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non limiting range for a therapeutically effective amountof an 161P2F10B ADC administered in combination according to theinvention is about 0.5 to about 10 mg/kg, about 1 to about 5 mg/kg, atleast 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, or at least 4 mg/kg.Other exemplary non-limiting ranges are for example about 0.5 to about 5mg/kg, or for example about 0.8 to about 5 mg/kg, or for example about 1to about 7.5 mg/kg. The high dose embodiment of the invention relates toa dosage of more than 10 mg/kg. It is to be noted that dosage values mayvary with the type and severity of the condition to be alleviated, andmay include single or multiple doses. It is to be further understoodthat for any particular subject, specific dosage regimens should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions, and that dosage ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition.

Clinical Development Plan (CDP)

The CDP follows and develops treatments of 161P2F10B ADCs in connectionwith adjunctive therapy or monotherapy. Initially, Pre-clinicaltoxicology studies are performed in non-human subjects (e.g., mice,monkeys, etc.) using standard protocols known in the art. TheH16-7.8mcMMAF was demonstrated to be well-tolerated in the non-humantoxicology studies. The human Clinical Trials initially demonstratesafety and thereafter confirm efficacy in repeat doses. Trials are openlabel comparing standard chemotherapy with standard therapy plus161P2F10B ADCs. As will be appreciated, one non-limiting criteria thatcan be utilized in connection with enrollment of patients is 161P2F10Bexpression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 161P2F10B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. 161P2F10B MAbs are found to be safe upon humanadministration.

Example 9 Antibody Drug Conjugation of H16-7.8 MAb Characterization

I) Peptide Mapping by Mass Spectrometry

Peptide mapping analysis was conducted. This method is used to confirmthe identity of H16-7.8mcMMAF and distinguishes native antibody(H16-7.8). The obtained H16-7.8mcMMAF and H16-7.8 were treated withdithiothreitol (DTT) to reduce disulfide bonds, followed by alkylationof the resulting free cysteines. Guanidine was used in this step toensure complete denaturation of the protein. After dialysis to removethe guanidine, the samples were digested with a specific endoproteinase,Lys-C. Lys-C cleaves peptide bonds on the C-terminal side of lysineresidues. The resulting peptides were analyzed by reversed phasechromatography coupled to mass spectrometry. The reversed phaseretention times and the observed mass to charge ratios of the peaks werecompared between H16-7.8mcMMAF and H16-7.8. LC-MS (liquidchromatography-mass spectrometry) analysis was carried out using aWATERS Acquity UPLC coupled to a WATERS Q-TOFp mass spectrometer. Thedigested sample was applied to YMC C18 column and eluted with anacetonitrile gradient containing trifluoroacetic acid. Representativepeptide maps for H16-7.8mcMMAF and H16-7.8 are shown in FIG. 14.

All three chromatograms in FIG. 14 seems to be identical except for thepeaks indicated by asterisk and arrow. As can be seen in the Figure,peak intensities indicated by asterisk were reduced in the conjugatedantibody compared to the native antibody. The peaks marked with an arrowrepresent new peaks that appeared on the conjugated antibody peptidemap. Specifically, the peaks marked with either an asterisk or with anarrow are believed to be a peptide destined for conjugation and theresulting conjugated peptide, respectively. FIG. 15 shows a portion ofthe mass spectra of the peak marked with an asterisk. The mass value ofthe signal that changed during conjugation is indicated by the “plus”sign. This peptide with an approximate m/z of 970.4 (+3 charge state)was identified as C225-K250 that originated from the hinge region of theheavy chain and contains the expected conjugation sites.

In order to identify the newly appeared peaks which are believed to beconjugated peptide in FIG. 14, LC-MS analysis was conducted using theelevated-energy (MSE) data acquisition technique. FIG. 16 shows theextracted ion chromatograms (XIC) for peptide maps of H16-7.8mcMMAF andH16-7.8 using the m/z of 619.4. This ion corresponds to a fragment ionof the drug moiety. Peaks observed in XIC at 619.4 are almost identicalto the peaks marked with an arrow in FIG. 14. Furthermore, no such peakswere detected in the chromatogram of the native antibody. Theseobservations suggest that the detected peaks in the XIC at m/z of 619.4were apparently drug conjugated peptides and are identified by itsintact mass values. The result was summarized in Table V. These resultssuggest that in case of the conjugate, predominant peptides are thoseconjugated to 2 drugs on the hinge region of heavy chain. These data areconsistent with the data obtained by the other orthogonal such as a DARanalysis.

II) Intact Mass Analysis by LC-MS

The full mass of the deglycosylated H16-7.8mcMMAF was determined byelectrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Thistechnique provides direct information about the drug-to-antibody ratio(DAR) value. Test samples were diluted by 250 mM sodium phosphatebuffer, pH 7.5 and then incubated overnight at 37° C. withGlycopeptidase F. The samples were injected onto a PLRPTM column (VarianTechnology), equilibrated at 90° C., and eluted with anacetonitrile/water gradient. The sample peaks were analyzed by anAcquity UPLC system coupled to an WATERS Synapt mass spectrometer(Waters) and masses were reconstructed from the raw data by an MaxEnt1software. An example mass spectral profile for the deglycosylatedH16-7.8mcMMAF is shown in FIG. 17. The predominant drug conjugatedantibody was a 4-drug loading species. This observation including anabundance of the unconjugated antibody in H16-7.8mcMMAF was consistentwith the results obtained by the other orthogonal methods, such as DARby RP-HPLC, peptide mapping and HIC assay.

III) Drug to Antibody Ratio (DAR) Analysis by RP-HPLC

Drug to Antibody Ratio (DAR) analysis was conducted for quantitativeHPLC determination of the relative amount of drug loading in each Lightchain and Heavy chain. DAR analyses were carried out using a PLRP-Sanalytical column, 2.1 mm×50 mm, with mobile phase A consisting of 2.0%formic acid and mobile phase B consisting of 2.0% formic acid plus 90%acetonitrile. For sample preparation, the drug conjugated antibody wascompletely reduced by DTT and then separated to the L chain, the drugconjugated L chain, the H chain and the drug conjugated H chains basedon the drug loading amount. 50 μg of sample was eluted using a flow rateof 0.5 ml/min, with detection at 280 nm. The molar ratio of drug toantibody ratio (DAR) is defined by the following equation.

${D\; A\; R} = {\left( {{\sum\limits_{n = 0}^{1}\left( {\frac{A\; U\; C_{{Light},n}}{A\; U\; C_{{Total},{Light}}} \times n} \right)} + {\sum\limits_{n = 0}^{5}\left( {\frac{A\; U\; C_{{Heavy},n}}{A\; U\; C_{{Total},{Heavy}}} \times n} \right)}} \right) \times 2}$

Where

DAR—drug to antibody molar ratio

n—number of mcMMAF drugs per Ab chain

AUCLight,n, AUCHeavy,n—area under curve for the light or heavy antibodychain with n drugs, respectively;

AUCTotal, Light(Heavy)—peak area under curve of the light or heavychain.

This method has been qualified using material from H16-7.8mcMMAF.Parameters evaluated included specificity, accuracy, repeatability,intermediate precision. A representative DAR profile for H16-7.8mcMMAFis shown in FIG. 18. DAR value is 4.0. Sample was subjected to LC-MSanalysis using same HPLC conditions of this method to identify theobserved peak. Results are summarized in Table VI. The peakidentification of the DAR results obtained during the qualification ofthis method has been confirmed orthogonally by LC-MS.

IV) Binding Affinity

H16-7.8 and H16-7.8mcMMAF were tested for their binding affinity to161P2F10B expressed on KU812 cells (human chronic myelogenous leukemiacells, ATCC). Briefly, twelve (12) dilutions of H16-7.8 or H16-7.8mcMMAFwere incubated with KU812 cells (50,000 cells per well) overnight at 4°C. at a final concentration of 160 nM to 0.004 nM. At the end of theincubation, cells are washed and incubated with anti-hIgG-PE detectionantibody for 45 min at 4° C. After washing the unbound detectionantibodies, the cells are analyzed by FACS.

MFI values were entered into Graphpad Prisim software and analyzed usingthe one site binding (hyperbola) equation of Y=Bmax*X/(Kd+X) to generateH16-7.8 and H16-7.8mcMMAF saturation curves. Bmax is the MFI value atmaximal binding of H16-7.8 or H16-7.8mcMMAF to KU812; Kd is H16-7.8 orH16-7.8mcMMAF binding affinity which are the concentration of H16-7.8 orH16-7.8mcMMAF required to reach halfmaximal binding. The calculatedaffinity (Kd) of H16-7.8 and H16-7.8mcMMAF is 0.08 nM and 0.25 nM on161P2F10B expressed on the surface of KU812 cells, respectively. (n=4).

V) Cytotoxicity

The H16-7.8, H16-7.8mcMMAF and a negative control ADC were separatelyserially diluted and added to a 96-well plate containing KU812 cells,which endogenously express 161P2F10B on the cell surface. After six daysof incubation, Alamar Blue® reagent is added to the antibody-cellmixture. AlamarBlue® is a cell viability indicator that uses the naturalreducing power of living cells to convert resazurin to the fluorescentmolecule, resorufin. Resazurin is reduced to resorufin, which producesvery bright red fluorescence. Viable cells continuously convertresazurin to resorufin, thereby generating a quantitative measure ofviability—and cytotoxicity. The percent cytotoxicity of H16-7.8mcMMAF isevaluated using fluorescence units obtained spectrophotometrically usingthe Synergy 4 Hybrid Multi-Mode Microplate reader (540/35, 620/40 nm).The linear range of the assay is approximately 3.9 to 1000 ng/ml. Thereare 9 points in the standard curve: the highest concentration ofH16-7.8mcMMAF is 1000 ng/ml followed by eight serial 1 to 2 dilutionsand a blank (0).

Calculate % Survival using formula below:

% Survival=(X−Blank)/(No treated−Blank)×100

Specific cytotoxicity activity of H16-7.8mcMMAF on KU812 cells: IC500.15 nM.

H16-7.8 and ADC control (antibody(non-anti-161P2F10B antibody)-mcMMAF)did not have cytotoxicity activity on KU812 cells.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables

TABLE I Tissues that express 161P2F10B when malignant. Kidney Colon LungOvary Breast Lymphoma Bone Uterus Pancreas Liver Prostate

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix A C D E F G H I K L M N P Q R ST V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3−4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1−4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3−2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2−2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3−3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2−2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2−3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins.

TABLE IV FACS MFI Values on Ku812 cells nM H16-7.8 H16-7.8mcMMAF 160 99108 80 92 102 40 98 108 20 89 97 10 75 89 5 71 80 2.5 65 68 1.3 59 600.63 57 57 0.31 58 54 0.16 53 47 0.078 47 37 0.039 36 30 0.020 27 200.010 18 14 0.0049 13 11 0.0024 9 8 0.0012 7 7 0.0006 6 6 0.0003 6 60.0002 6 5 0.0001 5 5

TABLE VSummary of the peak identification results on the drug conjugated peptides,potential conjugation sites (cystein residues) are set forth in bold type and underlined.Peak Predominant Calculate Sequence No. observed mass massTentative Identification Identification 1 1754.81 1736.51 S₂₀₈FNRGE C₂₁₄ + 1mcMMAF + H₂O, Light chain SEQ ID NO: 9 2 1736.82 1736.51S₂₀₈FNRGE C ₂₁₄ + 1mcMMAF, Light chain SEQ ID NO: 9 3 4679.29 4643.72 C₂₂₅ C VE C PP C PAPPVGPSVFLFPPKPK₂₄₉ + 2mc SEQ ID NO: 10 MMAF +2H₂0, Heavy chain 4 4661.29 4643.72 C ₂₂₅ C VE C PP CPAPPVGPSVFLFPPKPK₂₄₉ + 2mc SEQ ID NO: 10 MMAF + H₂0, Heavy chain 54643.20 4643.72 C ₂₂₅ C VE C PP C PAPPVGPSVFLEPPKPK₂₄₉ + 2mcSEQ ID NO: 10 MMAF, Heavy chain 6 4704.39 4643.72 C ₂₂₅ C VE C PP CPAPPVGPSVFLEPPKPK₂₄₉ + 2mc SEQ ID NO: 10 MMAF, Heavy chain 7 4644.384643.72 C ₂₂₅ C VE C PP C PAPPVGPSVFLFPPKPK₂₄₉ + 2mc SEQ ID NO: 10MMAF, Heavy chain 8 4626.27 4643.72 C ₂₂₅ C VE C PP CPAPPVGPSVFLFPPKPK₂₄₉ + 2mc SEQ ID NO: 10 MMAF-H₂O, Heavy chain

TABLE VI Peak identification results of DAR analysis by LC-MS PeakObserved Mass difference from No mass unconjugated peak Assignments L023596.1055 N/A Unconjugated L chain L1 24521.7031 925.6 1 drugconjugated L chain H0 50304.9102 N/A Unconjugated H chain H1 51230.3984925.5 1 drug conjugated H chain H2 52155.9023 1851.0 2 drug conjugated Hchain H3 53085.2813 2780.4 3 drug conjugated H chain H4 54006.74223701.8 4 drug conjugated H chain H5 54929.6002 4624.7 5 drug conjugatedH chain

TABLE VII Synthetic Scheme of mcMMAF

1. An antibody drug conjugate, comprising an antibody or antigen bindingfragment thereof that binds specifically to a 161P2F10B proteincomprising the amino acid sequence of SEQ ID NO:2, wherein the antibodycomprises the amino acid sequence of the V_(H) region of SEQ ID NO:7,from 20 to 142, and the V_(L) region of SEQ ID NO:8, from 20 to 127 andwherein said antibody is conjugated to monomethyl auristatin F (MMAF).2. The antibody drug conjugate of claim 1, wherein the antigen bindingfragment is an Fab, F(ab′)₂ or Fv fragment.
 3. The antibody drugconjugate of claim 1, wherein the antibody is a fully human antibody. 4.The antibody drug conjugate of claim 1, which is recombinantly produced.5. A pharmaceutical composition that comprises the antibody drugconjugate of claim 1 in a human unit dose form.
 6. The pharmaceuticalcomposition of claim 5, wherein the composition is for cancer treatment.7. The pharmaceutical composition of claim 6, wherein the cancer isrenal cancer or liver cancer.
 8. A method of inhibiting growth of cancercells in a subject, comprising: administering to said subject anantibody drug conjugate of claim
 1. 9. A method of delivering acytotoxic agent or a diagnostic agent to a cell, comprising: providingMMAF(s) conjugated to an antibody or fragment thereof that bindsspecifically to a 161P2F10B protein comprising the amino acid sequenceof SEQ ID NO:2, wherein the antibody comprises the amino acid sequenceof the V_(H) region of SEQ ID NO:7, from 20 to 142 and the V_(L) regionof SEQ ID NO:8, from 20 to 127, to form an antibody drug or fragmentdrug conjugate; and exposing the cell to the antibody drug or fragmentdrug conjugate.
 10. A method for treating tumor in a mammal, comprisingtreating the mammal with an effective amount of an antibody drugconjugate of claim
 1. 11. A method for reducing tumor growth in amammal, comprising treating the mammal with an effective amount of acombination of an antibody drug conjugate of claim 1 and radiation. 12.A method for reducing tumor growth in a mammal, comprising treating themammal with an effective amount of a combination of an antibody drugconjugate of claim 1 and a chemotherapeutic agent.
 13. A method forreducing tumor growth in a mammal, comprising treating the mammal withan effective amount of a combination of an antibody drug conjugate ofclaim 1 and a drug or biologically active therapy.
 14. A method fortreating cancer in a mammal, comprising treating the mammal with aneffective amount of a combination of an antibody drug conjugate of claim1 and a chemotherapeutic agent.